Methods for diagnosis and/or treatment of antiphospholipid antibody-related diseases and devices thereof

ABSTRACT

Methods for detecting anti-lipidic particles antibodies and lipidic particles in cellular membranes for the diagnosis of diseases associated to the antiphospholipid syndrome are disclosed. Kits or sets to put these methods of diagnosis into practice are disclosed. Methods for the therapeutically treatment of diseases associated to the antiphospholipid syndrome are disclosed as well. In addition, methods for the detection of the diverse physiologic states of cells, and those kits useful for this are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/632,735, filed Aug. 4, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates to obtaining antibodies recognizinglipids and more particularly, is related to methods for obtainingantibodies against lipidic structures different from the lipidicbilayer, and to the use of these antibodies in diagnostic and/ortreatment of diseases associated with the antiphospholipid syndrome; aswell as for the determination of physiological states of the cell.

BACKGROUND OF THE INVENTION

[0003] Considering the state of the art there are different studies inwhich evidence of the existence of antibodies that recognize lipids canbe found. For example, they have been detected in the serum of patientswith antiphospholipid syndrome, as was described by Asherson et al. intheir book “The antiphospholipid syndrome” in 1996 (CRC Press, BocaRaton). In the same way, antiphospholipid antibodies have been obtainedfrom animals that were experimentally treated with lipids by activeimmunization, in accordance with Alving in 1992 (Biochim. Biophys. Acta1113:307-322) or, in animals that received antiphospholipid antibodiesby passive immunization, as Tincani and Shoenfeld described in 1996 inthe above mentioned book.

[0004] The anti-lipid antibodies have been classified into two majorsubgroups according with the method used for their determination. Thesegroups are anti-cardiolipin antibodies and anticoagulant antibodies(Guglielmone y Fernandez, 1998, J. Rheumatol. 26:86-90).

[0005] The anti-cardiolipin antibodies are determined by methods inwhich cardiolipin immobilized in a solid phase is used. This wasdescribed by Harris et al. in 1985 (Clin. Rheum. Dis. 11:591-609), suchas the enzyme-linked immunosorbent assays and the radioimmunoassaysbetter known by their respective initial abbreviations as ELISA and RIAwhich have been broadly used in the above mentioned technique.

[0006] The anticoagulant antibodies are detected by methods in which theprolongation in the coagulation time of plasma samples is measured invitro, according with Bevers et al. 1991(Thromb. Haemost. 66:629-632).Some of these methods are: activated partial thromboplastin time (APTT),dilute Russell's viper venom time (dRVVT), protein C, and protein Samong others. In these methods, the anticoagulant antibodies are boundto phosphatidylethanolamine or to phosphatidylserine which areintermediary factors in the blood coagulation cascade, and when theconcentration of these lipids decrease due to the immune reaction, thecoagulation time is prolongated.

[0007] The anti-cardiolipin antibodies have the disadvantage ofproducing crossed reaction with other anionic lipids such asphosphatidylserine and phosphatidylglycerol. Due to the lack ofspecificity for a certain type of lipid, the above mentioned antibodiesare generally known as antiphospholipid antibodies.

[0008] In addition, antibodies against phosphatidylethanolamine havebeen detected in the sera from patients with antiphospholipid syndrome.Also, antibodies against phosphatidylcholine are detected in patientswith hemolytic anemia, as was described by Sugi and McIntyre (Blood86:3083-3089) and Arvieux et al. (Thromb. Haemost. 74:1120-1125),respectively, in 1995.

[0009] On the other hand, some studies have demonstrated that thebinding of antiphospholipid antibodies to the lipidic antigen increasesin the presence of a plasmatic protein. For example, in 1990, McNeil etal., determined that the binding of antibodies to cardiolipin wasmarkedly enhanced by the plasma protein β₂-glycoprotein I or apoproteinH (Proc. Nat. Acad. Sci. USA 87:4120-4124). Additionally, someanti-cardiolipin antibodies are bound directly to β₂-glycoprotein I, aswas described by Roubey et al. in 1995 J. Immunol. 154:954-960). Thesefindings suggest that the anti-cardiolipin antibodies may recognizeeither a cryptic epitope on β₂-glycoprotein I exposed on the complex ofβ₂-glycoprotein I-cardiolipin, or β₂-glycoprotein I alone but with avery low affinity towards the glycoprotein, as was described by Pengo etal. (1995, Thromb. Haemost. 73:29-34).

[0010] In accordance with these studies, it may be concluded that thebinding of antiphospholipid antibodies to lipidic antigens is alsoassociated with proteins. Sugi and McIntyre (op. cit., 1995) found thatthe proteins called kininogens are involved in the binding of antibodiesto phosphatidylethanolamine, whereas the proteins that are bound tophosphatidylserine, such as prothrombin, protein C, protein S andannexin V, have been implicated in the binding of anticoagulantantibodies to phosphatidylserine, according with the studies in 1994 byNakamura et al. (Biochim. Biophys. Res. Commun. 205:1488-1493) and byRoubey (Blood 84:2854-2867).

[0011] These studies indicate that the antigen of some antiphospholipidantibodies is really a complex formed by phospholipids and specificplasma proteins, but these proteins differ from those required forreactivity of antiphospholipid antibodies with cardiolipin.Nevertheless, in other studies, antiphospholipid antibodies that bounddirectly to the phospholipid have been identificated such as theanti-cardiolipin antibodies that do not require the β₂-glycoprotein I.Such studies were carried out by McNeil et al. in 1989 (Br. J. Haematol.73:506-513) and by Pengo and Basiolo in 1993 (Thromb. Res. 72:423-430).

[0012] On the other hand, some anti-cardiolipin antibodies, purified byaffinity chromatography, do not show anticoagulant activity (McNeil etal., op. cit., 1989; Shi et al., 1993, Blood 81:1255-1262). However,other studies demonstrated that the anti-cardiolipin and theanticoagulant antibodies were removed by adsorption with cardiolipin(Pengo and Biasiolo, op. cit., 1993; Pierangeli et al. 1993, Br. J.Haematol. 85:124-132).

[0013] Additionally, during studies in experimental animals, treated bypassive or active immunization, the employed methods for the detectionof antiphospholipid antibodies are the same as those described for thedetection of human antiphospholipid antibodies. Furthermore, in theseanimal models, the different organs and tissues were analized byanatomical and histopathological studies, by immunofluorescent studies,and even by fetal resorption analysis and consequently the producedlessions in fetuses and placentas of the female animal models were alsoanalyzed. These works were performed by Tincani y Shoenfeld (op. cit.1996) and by Shoenfeld and Ziporen (Lupus 7:S158-S161, 1998).

[0014] The previously mentioned studies, show that the antiphospholipidantibodies described in human patients and in animal models have a broadspecificity towards the lipidic antigens. This broad specificity of theantibodies may be attributed, among other causes, to the lack ofspecificity of the methods used for the detection of the above describedantibodies.

[0015] In such methods, it has not been considered the chemicalstructure and the molecular association of lipidic antigens, as well asthe chemical properties that the lipidic antigens have in the nature. Asa consequence, in the lipidic antigens that have been used in thosemethods, the phospholipids are bound to artificial solid supports, suchas in the ELISA and RIA methods, or they are in a molecular associationthat is not completely characterized, like in tests where theprolongation in the coagulation time is detected.

[0016] There are only a few studies in which the molecular structure ofthe phospholipid employed as antigen has been considered. For example,the reports of Rauch et al. in 1989 and in 1998 (Thromb. Haemost.62:892-896 and Thromb. Haemost. 80:936-941, respectively) and that ofBerard et al. U. Lab. Clin. Med., 1993, 122:601-605). In these reports,the authors demonstrated that the sera from some patients with systemiclupus erythematosus is inhibited in its anticoagulant activity byphosphatidylethanolamine associated in the hexagonal tubular II phase.This inhibition was not observed when the phospholipid was associatedinto the bilayer phase. However, the properties of the cellular membranecan not be related with the tubular association of phospholipids becausethis tubular lipidic association is practically incompatible with thevesicular structure of the cellular membrane, as different authors haveestablished. In other words, in the lipidic antigens used in thesestudies the phospholipids are in molecular arrangements that do notcorrespond to the molecular arrangements that they present in thecellular membrane.

[0017] Additionally, it is well-known that the molecular structure ofthe plasmatic membrane of mammal cells is like an associationheteropolymer formed by phospholipids, glycolipids, cholesterol,proteins and glycoproteins where the lipids are mainly in a moleculararrangement of bilayer. Nevertheless, it is also known that lipids mayhave molecular arrangements different to the bilayer and that sucharrangements depend on the molecular geometry of the lipids and thesurrounding conditions.

[0018] Cylindrical shaped lipids, such as phosphatidate,phosphatidylglycerol, phosphatidylinositol, phosphatidylcholine,phosphatidylserine, cardiolipin, sphingomyelin anddiglucosyldiacylglycerides, in an aqueous media are associated in closedbilayers, or liposomes. Cylindrical lipids constitute from 60 to 70% ofthe membranal lipids.

[0019] On the other hand, the conic shaped lipids such asphosphatidylethanolamine, monoglucosyldiacylglycerides, anddiacylglycerols, as well as the above mentioned lipids: phosphatidate,cardiolipin, phosphatidylserine, and phosphatidylglycerol but in thepresence of divalent cations are assembled in the molecular phase knownas hexagonal II (HII), which corresponds to tubular cylinders packedhexagonally. While the inverted cone shaped lipids, such aslysophospholipids and gangliosides are associated in micelles. Conic andinverted conic shaped lipids represent from 30 to 40% of the membranallipids.

[0020] Lipidic arrangements in hexagonal II or micellar phases, as wellas any other structural arrangement of lipids that do not form a bilayerbut that is immerse in a bilayer, are considered, for the purposes ofthis invention, as lipidic structures different to the lipidic bilayeror “lipidic particles”, independently of the kind of lipids that areforming these structures.

[0021] In the same way, it is known that in the presence of divalentcations, drugs like chlorpromazine and procainamide, non-polar peptides,proteins such as the protein of the bacteriophage M13, cholesterol,lanthanum ions, as well as changes in temperature and in the pH, theconic lipids form molecular arrangements different to the lipidicbilayer. These lipidic arrangements are of transient nature because whenthe concentration of the compounds that induced their formationdiminishes or when the temperature or the pH changes again, the conicshaped lipids return to the bilayer arrangement as was described byCullis et al., in 1991 (Membrane Fusion. Marcel Dekker, N.Y.), by Baezaet al. in 1995 (Biochem. Cell Biol. 73:289-297) and Aguilar et al., in1999 (J. Biol. Chem. 274:25193-25196). Lipidic bilayer moleculararrangements are observed like a smooth surface by cryofractureanalysis.

[0022] Lipids in general are molecules with low immunogenicity, and ofthe two molecular arrangements that the lipids may adopt in cellularmembranes, it is considered that the lipidic bilayer will be the lessimmunogenic because it is the one that mainly constitutes the matrix ofall cellular membranes.

[0023] However, it is known that the lipidic structures different to thebilayer, which are stabilized with divalent cations and that areobserved as protuberances on the smooth surface of the bilayer bycryofracture analysis, induce the formation of antibodies that recognizethe lipids that are associated in lipidic particles and they do notreact with lipids associated in bilayer.

[0024] In connection with the above-mentioned studies, Baeza and theircollaborators in 1995 (op. cit.) reported the elaboration of liposomeswith lipid molecular arrangements different to the bilayer, as well asthe antigenic activity of these molecular arrangements, because theywere able to obtain polyclonal antibodies with them. By means ofcytofluorometric analysis of the immune reaction they were also able toidentify the presence of lipidic structures in the liposomes described,using for it anti-lipidic particles polyclonal antibodies obtained frommice sera.

[0025] To this respect, the mice were immunized by the introduction ofartificially formed lipidic particles which when are present in excesscaused the wanted immune reaction. Until now, it is believed thatmolecular arrangements different to the bilayer or lipidic particleswould be also scarce immunogenics when they are present in the nature,for example in cells of human and animals, because lipidic particles aretransient and therefore they would not be detected by immune systems.

[0026] Additionally, from the analysis of the above mentioned studies,one can observe that the cardiolipin is the only lipid that has beenable to react with antibodies present in patients with theantiphospholipid syndrome or associated illnesses, and that the otherphospholipids usually present in the cellular membrane in generalrequire to be associated with proteins to react with the antibodies fromthese patients, or, they require to be associate in a moleculararrangement incompatible with the molecular structure of the cellularmembrane; with the exception of the studies of Baeza and theircollaborators (op. cit. 1995) on the anti-lipidic particles antibodieswhich react with a lipidic molecular arrangement similar to the one thathas been described in cellular membranes.

[0027] To this respect, the presence in sera from patients with theantiphospholipids syndrome of anti-cardiolipin antibodies, amitochondrial lipid, of anti-nuclear antibodies and of anti-DNAantibodies, it is indicative of the existence of previous events thatcause immunologic damage to cellular membranes, with the disruption ofthe cells and the exhibition of the intracellular components to theimmunologic system, causing the corresponding immunologic reaction thatcontributes to the development of the syndrome. However, up to now therehave not been found studies which allow to determine the events thatcause the disruption of the cellular membrane. In other words, with theexistent knowledge so far it is impossible to detect theanti-cardiolipin antibodies, the anti-nuclear or even the anti-DNAantibodies before the damage that has been caused to the cell, impedingan early diagnosis and treatment of the illnesses associated with thesyndrome.

[0028] Additionally, in the Doctoral Thesis presented by LeopoldoAguilar in Dec. 17, 1997 (Determination of non-bilayer lipidicarrangements in liposomes and cellular membranes with monoclonalantibodies”, Doctoral Thesis, National School of Biological Sciences,National Polytechnic Institute, México) 5 sera from patients withprimary antiphospholipid syndrome and 5 sera from patients with systemiclupus erythematosus were analyzed, these illnesses were corroborated byclinical characteristics that the patients presented and by means of thedetection of anti-cardiolipin antibodies, and of anti-nuclearantibodies, these last ones in the case of the sick persons with lupus.The analyzed sera from all patients also presented anti-lipidicparticles antibodies, detected according to the techniques ofliposomal-ELISA and of liposomal cytofluorometry described in the abovementioned Thesis.

[0029] This discovery, however, does not show any advantage for theearly detection of the illnesses, since the presence of theantiphospholipid antibodies and of the anti-lipidic particles antibodiesin those patients can be explained according with two hypothesis.

[0030] The first one, assumes that an unknown factor causes thedestruction of the cellular membrane, which promotes the formation oflipidic particles from the membranal lipids that enter in contact withthe immunologic system together with the intracellular components, withthe consequent simultaneous formation of anti-lipidic particlesantibodies, and anti-cardiolipin and anti-nuclear antibodies.

[0031] The second hypothesis, consists on assuming that the lipidicparticles are formed in the cellular membrane before its destruction,and they would form anti-lipidic particles antibodies that would destroythe membrane, exposing the intracellular components to the immunologicsystem and giving place later on to the formation of anti-cardiolipinand anti-nuclear antibodies.

[0032] This second hypothesis was proposed in the Master Thesispresented by Monica Lara on Aug. 20, 1999 (“Detection of anti-lipidicparticles antibodies in patients with the anti-phospholipid syndrome,”Master Thesis, Escuela Nacional de Ciencias Biológicas [National Schoolof Biological Sciences], Instituto Politécnico Nacional [NationalPolytechnic Institute], Mexico.

[0033] So far, none of the two hypothesis has been demonstrated, whichis of supreme importance for the treatment of the illnesses, sinceshould the second hypothesis probed to be certain, it would be possibleto detect the illnesses above mentioned in their early stages, and also,it would be possible the prevention, cure or patient's improvement fromsuch illnesses.

[0034] Derived from the above-mentioned hypothesis, it has been aimed tosuppress the inconveniences of the induction and detection ofantiphospholipid antibodies techniques caused by the structure andmolecular association of the antigens used in these methods, by theemployment of lipidic antigens with a structure and molecularassociation similar to the one found in patients with illnessesassociated with antiphospholipids antibodies. These novel lipidicantigens have been used for the induction and detection of anti-lipidicparticles antibodies that allow an early diagnosis of these illnesses,as well as for the determination of physiologic states of the cell, asapoptosis, or programmed cellular death (Pittoni and Isenberg, 1998,Semin. Arthritis. Rheum. 28:163-178) and those which are present in thecellular cycle (Go, G1, G2 and M) among others.

OBJECTS OF THE INVENTION

[0035] Keeping in mind the deficiencies in the structure and in themolecular association of the antigens that are used in the techniques ofinduction and detection of antiphospholipid antibodies from the methodsof the previous techniques, one of the objectives of the presentinvention consists on using lipidic antigens with a structure andmolecular association similar to the one that is present in patientswith illnesses associated with antiphospholipid antibodies, with thepurpose of providing a method for the detection of anti-lipidicparticles antibodies.

[0036] It is another objective of the present invention, to provide adiagnosis method which uses monoclonal antibodies specific to lipidicantigens that respond in the same way that the anti-lipidic particlesantibodies present in sera from patients with diverse illnessesassociated with antiphospholipid antibodies, with the purpose ofdesigning a strategy for the treatment of these patients against suchillnesses.

[0037] It is an additional objective of the present invention, toprovide a kit or diagnosis set for the detection of anti-lipidicparticles antibodies in early stages of illnesses that present suchantibodies in animals and in humans.

[0038] It is another objective of the present invention, to provide akit or diagnosis set for the detection of lipidic particles in themembranes of the cells of ill entities, human or animal, that presentanti-lipidic particles antibodies.

[0039] It is still another objective of the present invention, toprovide a method for the prevention, cure or patient's improvement fromsuch illnesses by means of the inhibition or the blockage ofanti-lipidic particles antibodies.

[0040] Another objective of the present invention still consists onproviding a method for the prevention, cure or patient's improvementfrom such illnesses, by means of the stabilization of cellular membranesthat impedes the formation of lipidic particles and therefore the laterformation of anti-lipidic particles antibodies.

[0041] An additional objective of the present invention consists onproviding methods and its corresponding kits for the detection of thedifferent physiologic states that can present the cells, which can leadto the prevention of illnesses related with antiphospholipid antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 shows the analysis by the liposomal-ELISA method of thereaction between control sera from human healthy blood donators, or serafrom patients with the antiphospholipid syndrome, and liposomal antigensmade from egg-yolk phosphatidyl-choline:phosphatidate [PC:PA] (2:1 moleratio) bearing lipidic particles induced by calcium. Furthermore, thereaction of H308 monoclonal antibody with these antigens is alsoindicated.

[0043]FIGS. 2A and 2B illustrate the fluorescence graphs of liposomesmade from PC:PA (2:1 mole ratio), with and without lipidic particlesinduced by calcium, that were incubated with Tris-NaCl (10 mM, 1 mM) pH7, or with the second antibody conjugated to peroxidase.

[0044]FIGS. 3A-3I show the cytofluorometric analysis of the reactionbetween H308 monoclonal antibody and liposomal antigens made from PC:PA(2:1 mole ratio) or from dipalmitoylphosphatidylcholine:egg-yolkphosphatidylcholine:dipalmitoylphosphatidate [DPPC:PC:DPPA] (1.2:0.8:1mole ratio). As well as the cytofluorometric analysis of liposomalantigens from PC:PA (2:1 mole ratio) in Tris-NaCl (10 mM, 1 mM), with orwithout lipidic particles induced by calcium, in absence of the H308monoclonal antibody.

[0045]FIGS. 4A-4X show the cytofluorometric analysis of the reactionbetween sera from human healthy blood donators or from patients with theantiphospholipid syndrome, and liposomal antigens made from PC:PA (2:1mole ratio) bearing lipidic particles induced by calcium.

[0046]FIGS. 5A-5C show the cytofluorometric analysis which indicate thatthe AC15 serum from a patient with primary antiphospholipid syndromedoes not show any immunoreaction with liposomal antigens made fromDPPC:PC:DPPA (1.2:0.8:1 mole ratio) that lack lipidic particles.

[0047]FIG. 6 is a picture of C5337 human pancreas cancer cells thatshows the immunoreaction between H308 monoclonal antibody and lipidicparticles from the membranes of these cells.

[0048]FIG. 7 illustrates the analysis by the cellular-ELISA method ofthe reaction between sera from patients with the antiphospholipidsyndrome and C5337 human pancreas cancer cells; patients sera were usedadsorbed and without any adsorption with liposomal antigens made fromPC:PA (2:1 mole ratio) bearing lipidic particles induced by calcium.

[0049]FIGS. 8A-8F show the graphs of liposomal fluorescence andliposomal bilayers complexity which analyze the reaction betweenliposomal antigens and sera from BALB/c mice before or after they wereimmunized with PC:PA (2:1 mole ratio) liposomes bearing lipidicparticles induced by manganese. Liposomal antigens used inimmunoreactions were the same ones used for mice immunization.

[0050]FIGS. 9A-9F show the graphs of liposomal aggregation and liposomalbilayers complexity which analyze the reaction between liposomalantigens and sera from BALB/c mice before or after they were immunizedwith PC:PA (2:1 mole ratio) liposomes bearing lipidic particles inducedby manganese. Liposomal antigens used in immunoreactions were the sameones used for mice immunization.

[0051]FIGS. 10A-10F show the graphs of liposomal fluorescence andliposomal bilayers complexity which analyze the reaction betweenliposomal antigens and sera from BALB/c mice before or after they wereimmunized with PC:PA (2:1 mole ratio) liposomes bearing lipidicparticles induced by procainamide. Liposomal antigens used inimmunoreactions were the same ones used for mice immunization.

[0052]FIG. 11 is a photograph of a seven (7) month old BALB/c femalemouse immunized with PC:PA (2:1 mole ratio) liposomes bearing lipidicparticles induced by chlorpromazine, where alopecia and lesions on theface in the form of butterfly wings are observed.

[0053]FIGS. 12A-12I are the cytofluorometric analysis of the reactionbetween liposomal antigens and sera from BALB/c mice before or afterthey were treated by intramuscular injection each 24 hs during 2-monthswith 3 mg/Kg, of body weight, of the lipidic particles inducer drugprocainamide. Liposomal antigens used in immunoreactions were made fromPC:PA (2:1 mole ratio) bearing lipidic particles induced byprocainamide.

[0054]FIG. 13 illustrates outlines of the chemical structure ofphosphorylcholine, glycerolphosphorylcholine, phosphorylserine,glycerolphosphorylserine and phosphoryl-ethanolamine that are used ashaptens in the inhibition of anti-lipidic particles antibodies.

[0055]FIG. 14 illustrates the graphs of the inhibition of H308monoclonal antibody with phosphorylcholine, glycerolphosphorylcholine,phosphorylserine, glycerolphosphorylserine or phosphorylethanolaminehaptens. Furthermore, the reaction of H308 monoclonal antibody withliposomes made from phosphorylcholine is also showed.

[0056]FIG. 15 is a scheme of the lipids associated in the moleculararrangement of bilayer and of inverted micella which is inserted in anopen lipidic bilayer which constitute the lipidic particle on the whole.Arrows indicate the different molecular arrangements adopted by lipids.

[0057]FIGS. 16A-16H illustrate the graphs of liposomal aggregation andliposomal bilayers complexity that analyze the lipidic bilayersstabilization of liposomal antigens made from PC:PA (2:1 mole ratio)treated with the lipidic particles inducer drugs chlorpromazine orprocainamide and/or with the lipidic bilayer stabilizer drugschloroquine or spermidine.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMIENTS

[0058] Despite the transitory nature of lipidic particles in activecellular membranes, it has been found surprisingly that sera frompatients that present illnesses associated with the antiphospholipidsyndrome react with C5337 human pancreas cancer cells, which isindicative of a prolonged presence of lipidic particles in cellularmembranes of these patients.

[0059] Equally, when an antigen that contains lipidic particles isadministered in BALB/c mice it has been found surprisingly that thesemice developed alopecia and lesions on the face in the form of butterflywings, similar to those described in some human autoimmune illnesses, aswell as deposits of immune complexes and pathological alterations intheir different organs. Additionally, it has been also found that thesemice firstly developed anti-lipidic particles antibodies andsubsequently anti-cardiolipin antibodies, lupus anticoagulant andanti-nuclear antibodies, which confirms that anti-lipidic particlesantibodies constitute the first stage in the development of illnessesassociated with antiphospholipid antibodies.

[0060] According to the above-mentioned experiments, the presence ofanti-lipidic particles antibodies in one of the first stages of theillness, indicates that one of the first events that occurs in theantiphospholipid syndrome is the formation of anti-lipidic particlesantibodies. These antibodies when reacting with lipidic particles incellular membranes, cause damage in these membranes, and finally cellsdisruption and the exhibition of the intracellular components to theimmunitary system; which explains the subsequent presence ofanti-cardiolipin antibodies, where cardiolipin is a mitochondrial lipid,of anti-nuclear and anti-DNA antibodies, which have been reported aspresent in these illnesses in humans.

[0061] For the purposes of the present invention, we understand as“illness associated with antiphospholipid antibodies” to any illnessthat presents antiphospholipid antibodies in any development step. Someof such illnesses are mentioned next, in an enunciative fashion, but notlimitative: primary or secondary antiphospholipid syndrome; in the lastcase, associated with autoimmune illnesses such as vasculitis,rheumatoide arthritis and systemic lupus erythematosus; illnesses thatcause an increase in the cellular division, as they can be neoplasias ofthe type of carcinoma in liver or in ovary, lymphomas, leukemias ormyeloproliferative disorders; viral infections as mononucleosisinfectious and the acquired immunodeficiency syndrome; illnesses takenplace by bacteria, as syphilis; and, illnesses taken place by protozoaas malaria. Additionally, the presence of antiphospholipid antibodieshas been related with myocardial infarction and senility.

[0062] Therefore, an aspect of the present invention is to develop adiagnosis method for determining if an individual having clinicalcharacteristics of the primary antiphospholipid syndrome (Table 1), orone of the illnesses associated to the secondary antiphospholipidsyndrome (Table 1) and who does not present yet anti-cardiolipinantibodies, lupus anti-coagulant, anti-DNA or anti-nuclear antibodies,does have an illness associated to the presence of antiphospholipidantibodies; where such method comprises the steps of detecting in adirect or indirect fashion the presence or absence of lipidic particlesin a sample from said individual, and to observe whether lipidicparticles are detected or not, where the presence of said lipidicparticles indicates the development of an illness associated to thepresence of antiphospholipid antibodies in said individual.

[0063] In a preferred embodiment, the detection of lipidic particles iscarried out in an indirect fashion by means of the use of an antigencontaining lipidic particles reacting with the serum of the subject withthe purpose of determining if in this serum anti-lipidic particlesantibodies exist, such a determination being carried out preferably bymeans of the use of at least one technique selected from the groupconsisting of ELISA, cytofluorometry, and immunofluorescence.

[0064] In a specific embodiment, the antigen containing lipidicparticles is selected among neoplastic cells and liposomes whereliposomes are formed starting from at least one lipid susceptible tochange its molecular geometry by means of changes in temperature,presence of divalent cations, and/or drugs. This lipid being selectedpreferably among phosphatidate; cardiolipin; phosphatidylglycerol;phosphatidylinositol; diacylglycerol; sphingomyelin; phosphatidylserine;monoglucosyldiacylglyceride or phosphatidylethanolamine. In a favoritemodality, this lipid is found in abundance in cellular membranes.

[0065] In a specific embodiment, lipids used to form liposomes areselected according to their availability in cellular membranes andagainst which anti-lipid antibodies have been detected in humans, usingpreferably one lipid with a cylindrical molecular shape in combinationwith one lipid with a conical molecular shape in a mole ratio between1:1 to 4:1. In an additional modality, a combination ofphosphatidylcholine with phosphatidate from egg yolk in a 2:1 molarratio is used.

[0066] In another additional embodiment, at least an anti-lipidicparticles polyclonal or monoclonal antibody is made to react withneoplastic cells or liposomal antigens to confirm the presence or not ofanti-lipidic particles antibodies in the individual serum.

[0067] In another preferred embodiment, the detection of lipidicparticles is carried out in a direct fashion making react cells from thesubject with at least an anti-lipidic particles polyclonal or monoclonalantibody, preferably by means of the use of at least one techniqueselected from the group consisting of immunofluorescence,cytofluorometry and ELISA.

[0068] In an additional embodiment, besides cells of the subjects, it ismade to react with the anti-lipidic particles antibodies at least anantigen that contains lipidic particles, preferably selected betweenneoplastic cells and liposomes with at least one lipid susceptible tochange its molecular geometry by means of changes in temperature,presence of divalent cations, and/or drugs, this lipid being selectedpreferably among phosphatidate; cardiolipin; phosphatidylglycerol;phosphatidylinositol; diacylglycerol; sphingomyelin;phosphatidyl-serine; monoglucosyldiacylglyceride orphosphatidylethanolamine

[0069] In a specific embodiment, lipids used to form liposomes areselected according to their availability in cellular membranes andagainst which anti-lipid antibodies have been detected in humans, usingpreferably a cylindrical lipid in combination with a conical lipid in amole ratio between 1:1 to 4:1. In an additional modality, a combinationof phosphatidylcholine with phosphatidate from egg yolk in a 2:1 molarratio is used.

[0070] To obtain liposomes that are used in several modalities of thepresent invention, it is used preferably the reverse phase evaporationmethod, modified by Baeza and collaborators in 1994 J. Mol. Evol.,39:560-568), and subsequently liposomes are treated with a lipidicparticles inducer agent, preferably selected between divalent cationsand drugs, preferably those that produce the lupus induced by drugs inhumans, or combinations of the same ones; where the procedure to formlipidic particles is carried out preferably by means of incubation ofliposomes with an effective quantity of the lipidic particles induceragent at a temperature between 25 to 40° C., this effective quantitybeing preferably in a mole ratio lipids: lipidic particles inducer agentfrom 1:0.01 up to 1:300.

[0071] On the other hand, anti-lipidic particles polyclonal antibodiesuseful for diverse modalities of the present invention, are obtained byany known mice immunization method, using an antigen that containslipidic particles, preferably by means of a immunization procedure ofthe type described by Baeza and collaborators (op. cit., 1995), whichcomprises:

[0072] A) A first step of mice immunization using intrasplenic injectionof an effective dose of liposomes obtained from lipids against whichanti-lipid antibodies have been detected in humans, where theseliposomes contains lipidic particles in their surface.

[0073] B) A second step of mice immunization using intraperitonealinjection of the same liposomes and with the same doses used by thefirst immunization step.

[0074] When concluding these steps, immunized mice present anti-lipidicparticles polyclonal antibodies which can be detected by liposomal-ELISAmethod and/or liposomal cytofluorometry method.

[0075] In a specific embodiment, the effective liposomes doses were from50 to 200 mg, preferably incubated in a solution from 0.1 to 10 mM ofCaCl₂, MnCl₂, chlorpromazine, procainamide or combinations of the sameones in the presence of a buffer solution with pH between 7.0 to 7.4.

[0076] In an additional embodiment, in the first immunization step it isnecessary to administer liposomes at least 2-times by intrasplenicinjection with intervals of 1-week, and in the second immunization stepit is necessary to introduce liposomes by intraperitoneal injection atleast 4-times with intervals of 2-weeks, according to the methoddescribed by Nilsson et al. in 1987 (J. Immunol. Methods 99:67-75) andmodified by Aguilar in 1994 (Physical and immunologic characterizationof non-bilayer molecular arrangements in liposomes”, Master Thesis,National School of Biological Sciences, National Polytechnic Institute,México).

[0077] In another additional embodiment, mice used for immunization,were selected from singenic strain, using preferably 2-months age BALB/cfemale mice.

[0078] Starting from the immunized mice, it is possible to obtainmonoclonal antibodies by means of any well-known method, preferably bymeans of the obtention of a hybridoma. In a specific modality, thehybridoma was obtained according with the following steps:

[0079] A) Mice that were immunized by intrasplenic and intraperitonealinjections received a third immunization by intravenous administrationof the liposomes doses used for the first and second immunization steps.

[0080] B) A fusion step of immunized mouse spleen cells with myelomamouse cells that do not secrete gamma chains neither kappa chains. Thisfusion was carried out at least 4-days after the intravenousimmunization to obtain at least a hybridoma producing an anti-lipidicparticles monoclonal antibody.

[0081] C) A step of hybridomas selection, in which the hybridoma isselected among those which present detectable immunoreaction, using forthe detection of anti-lipidic particles antibodies the liposomal-ELISAmethod and/or the cytofluorometry method.

[0082] In a specific embodiment, the effective liposomes doses werebetween 50 to 200 mg, and preferably incubated with a solution from 0.1to 10 MM of CaCl₂, MnCl₂, chlorpromazine, procainamide or combinationsof the same ones in presence of a buffer solution with pH between 7.0 to7.4.

[0083] In an additional embodiment, the first immunization step includethe administration of liposomes at least 2-times by intrasplenicinjection with intervals of 1-week, and the second immunization stepinclude the introduction of liposomes by intraperitoneal injection atleast 4-times with intervals of 2-weeks, using the method described byNilsson et al. (op. cit., 1987) and modified by Aguilar (op. cit.,1994).

[0084] In another additional embodiment, mice used for immunization,were selected from singenic strain, using preferably 2-months age BALB/cfemale mice.

[0085] In a preferred embodiment of the present invention, immunizedmouse spleen cells were obtained according to the procedure described byAguilar (op. cit., 1997), by dispersion of the mouse spleen in anappropriate cellular culture medium, preferably incomplete DMEM mediumor RPMI medium added with glutamine 200 mM and glycine 100 mM, followedby diverse purification steps and of an erythrocytes lysis, preferablywith ammonium chloride, which only disrupted erythrocytes withoutaffecting lymphocytes neither leukocytes in general.

[0086] Cells of the P3X63Ag8U.1 cellular line of mouse myeloma obtainedby Yeltan (Curr. Top. Microbiol. Immunol., 1978, 81:1-7) are preferablyused. This cellular line is derived from the cellular line obtained byKohler and Milstein (Nature, 1975, 256:495-497) from BALB/c female miceMOPC21 myeloma.

[0087] Regarding the method used for cellular fusion, preferably the onedescribed by Aguilar (op. cit., 1997) is used, this method consists inusing the immunized mouse spleen and myeloma cells with a viabilityhigher than 95%, which are centrifuged and mixed in a cellularproportion 1:1, to be subsequently subjected to diverse washes steps andcultivated in cellular culture microtiter plates previously seeded withmacrophages.

[0088] With respect to the methods used for the detection ofanti-lipidic particles antibodies in patients sera by means of the useof antigens that contain lipidic particles, or for the detection oflipidic particles in cells from patients by using anti-lipidic particlesantibodies, the favorite techniques will be next described for suchdetections.

[0089] It is important to point out that in the description of thesetechniques the term “antibody porter” refers to any fluid susceptible ofcontaining anti-lipidic particles antibodies, as it can be a plasm or aserum from human or from animal origin, a solution or a suspension;while the term “antigen” refers to those structures susceptible ofcontaining lipidic particles such as liposomes or cells.

[0090] Additionally, it is also important to point out that in aspecific modality of the present invention, before start using anydetection method, an inactivation of sera or of plasms was carried outby increasing their temperature, preferably subjecting sera or plams attemperatures between 50 to 60° C. for 0.25 to 1 h.

[0091] On the other hand, the liposomal-ELISA method (Aguilar, op. cit.,1994; 1997; Aguilar et al., op. cit., 1999), as its name indicates, itis applicable in those cases in which the antigen is a liposome,independently of the origin of the antibody porter, and it comprises thefollowing steps:

[0092] A) A first step of addition and incubation, in which an effectivequantity of an antigen suspension is added to each one of the wells ofthe ELISA microtiter plate. This microtiter plate selected among thosewith a high lipidic antigens binding property and said microtiter plateis incubated between 25 to 30° C. for 0.25 to 2 h.

[0093] B) A second step of addition and incubation, in which aneffective quantity of a blocking solution is added to each one of thewells of the ELISA microtiter plate with a high lipidic antigens bindingproperty, and said microtiter plate being incubated at a temperaturebetween 25 to 30° C. for 0.25 to 2 h.

[0094] C) A step of elimination of blocking solution, preferably bysuction, with caution to avoid that microtiter plate becomes dry whenblocking solution is eliminated, because liposomal antigens can bedamaged.

[0095] D) A third step of addition and incubation, in which an effectivequantity of the antibody porter is quickly added, to avoid thatmicrotiter plate becomes dry, to each one of the wells using an antibodyporter dilution from 1:5 to 1:1000 into blocking solution; saidmicrotiter plate being incubated for 0.25 to 2 h at a temperaturebetween 25 to 30° C.

[0096] E) A first step of washing, in which the microtiter plate iswashed with the blocking solution, preferably repeating 4-times andavoiding that microtiter plate becomes dry when eliminating the blockingsolution.

[0097] F) A fourth step of addition and incubation, in which aneffective quantity of a second antibody is added to each one of thewells of microtiter plate. This plate is incubated in the darkness for0.25 to 2 h at a temperature between 25 to 30° C. Second antibody isselected preferably among antibodies from a different specie to that ofthe antibody porter and they can be anti-Fc of human IgG, IgA and IgM orof the animal in study, or antibodies anti-Fc of IgM or of IgG dependingon the nature of the monoclonal antibody when this is the antibodyporter. Second antibody is used at a final dilution into blockingsolution between 1:000 and 1:3500 and it is conjugated to an enzymepreferably to peroxidase.

[0098] G) A second step of washing, in which the microtiter plate iswashed with blocking solution, preferably repeating 4-times and avoidingthat microtiter plate becomes dry when eliminating the blockingsolution.

[0099] H) A fifth step of addition and incubation, in which an effectivequantity of the peroxidase substrates is added to each one of the wellsand said microtiter plate is incubated for 0.1 to 0.5 h at a temperaturebetween 35 and 40° C., stopping the peroxidase reaction by adding aneffective quantity of sulfuric acid.

[0100] I) A step of analysis, in which microtiter plate is analyzed in areading device for ELISA, preferably at 492 nm.

[0101] In a specific embodiment, the antigen suspension is obtained bysuspending liposomes in a buffer solution at pH between 7.0 to 7.4, in arelationship from 1 to 5 mmole of the antigen per liter of buffersolution.

[0102] The blocking solution, includes a buffer solution at pH between7.0 to 7.4, and at least a solution with a high content of proteins,preferably gelatin at 0.4%, weight by volume, with or without aneffective quantity of a lipidic particles inducer agent, preferably withthe effective quantity and the lipidic particles inducer agent used toform the antigen.

[0103] In a preferred embodiment, the effective quantity of the antigensuspension in the stage A it is of 50 to 100 μl. The second antibody canbe also conjugated to the enzyme alkaline phosphatase, instead ofperoxidase, in this case the corresponding alkaline phosphatasesustrates are used.

[0104] Liposomal-ELISA method allows the simultaneous determination ofanti-lipidic particles antibodies in at least 40 sera samples, each oneby duplicate, in a single microtiter plate, therefore, this method canbe easily applied to the diagnosis of illnesses where this type ofantibodies are present.

[0105] On the other hand, the liposomal cytofluorometry method (Baezaand collaborators., op. cit., 1995), as its name indicates, it isapplicable in those cases in which the antigen is a liposome,independently of the origin of the antibody porter, and it includes thefollowing steps:

[0106] A) A first step of addition and incubation, in which the antibodyporter is added to the antigen suspension. This antibody porter isdiluted from 1:5 to 1:1000 into a buffer solution at pH between 7.0 to7.4, and the resulting mixture is incubated for 0.25 to 2 h at atemperature between 35 and 40° C.

[0107] B) A first step of washing, in which the antigen bound to theantibody porter is washed with a buffer solution at pH between 7.0 to7.4, with or without an effective quantity of a lipidic particlesinducer agent, preferably with the same quantity and the same induceragent used to obtain the antigen.

[0108] C) A step of recovery, in which the antigen bound to the antibodyporter is recovered preferably by centrifugation.

[0109] D) A second step of addition and incubation, in which aneffective quantity of a second antibody is added to the antigen bound tothe antibody porter. The resulting mixture is incubated for 0.25 to 2 hin the darkness at a temperature between 35 to 40° C. Second antibody isselected preferably among antibodies from a different specie to that ofthe antibody porter and they can be anti-Fc of human IgG, IgA and IgM orof the animal in study, or antibodies anti-Fc of IgM or of IgG dependingon the nature of the monoclonal antibody when this is the antibodyporter. Second antibody is used at a final dilution from 1:25 to 1:500into a buffer solution at pH between 7.0 to 7.4, and it is conjugated toa substance or fluorescent substratum, preferably to fluoresceinisothiocyanate (FITC).

[0110] E) A second step of washing, in which the antigen bound to theantibody porter and to the second antibody is washed with a buffersolution at pH between 7.0 to 7.4, with or without an effective quantityof a lipidic particles inducer agent, preferably with the same quantityand the same inducer agent used to obtain the antigen.

[0111] F) A step of suspension and analysis, in which the antigen boundto the antibody porter and the second antibody is suspended in atransporting solution, selected preferably between FACS Flow (BecktonDickinson Co.) and Haema Line 2 (Serotono-Baker Diagnostics, INC) in arelationship from 1 to 5 mmole of the antigen in a liter of solution;this solution being preferably filtered previously with a 0.22 μmMillipore filter pore diameter, the obtained mixture being analyzed in aflow cytometer, preferably with a single 488 nm argon laser beam.

[0112] In a preferred embodiment, the antigen suspension is obtainedsuspending liposomes in a buffer solution at pH between 7.0 to 7.4, in arelationship of 1 to 5 mmole per liter of buffer solution. Furthermore,the fluorescent substrate can also be selected from the group consistingof phycoerythrin, Cy3 and Percp.

[0113] Liposomal cytofluorometry method has a sensibility 10-fold higherthan liposomal-ELISA method in the detection of anti-lipidic particlesantibodies. Therefore, this method must be applied when some doubtfulresult has been obtained with the liposomal-ELISA method.Cytofluorometry method also allows to analyze the presence of lipidicparticles in liposomal or cellular antigens, as well as to compare thedifferent types of reaction of polyclonal or monoclonal antibodies withthe lipidic particles of these antigens.

[0114] In another preferred embodiment of cytofluorometry method, thesuspension of the antigen can be from human or animal cells, and thesuspension of the antigen is obtained by suspending the cells,preferably isolated cells as erythrocytes, leukocytes and evenplaquettes in a buffer solution at pH between 7.0 to 7.4. With theexception of this difference regarding the antigen, the steps of thiscytofluorometry method are as those described in subparagraph (A) to (F)for liposomal cytofluorometry method in which liposomes are used asantigens.

[0115] Regarding cellular methods, the immunofluorescence method forcells, applicable when the antigen is a cell, comprises the followingsteps:

[0116] A) A step of cells culture, in which an effective quantity of theantigen is placed, preferably 1×10⁶ cells, in a micro covers glassinside each well of a cell culture plate and it is incubated under anatmosphere containing an effective CO₂ quantity at a temperature between35 to 40° C. until cellular confluence reaches 90%.

[0117] B) A first step of washing, in which the antigen is washed withan appropriate cell culture medium, preferably repeating 2-times, andwith a phosphates buffer solution at pH between 7.0 to 7.4, understerility conditions. Avoiding that the surface of cellular culturebecomes dry when eliminating the phosphates buffer solution, which candamage cellular antigens.

[0118] C) A first step of addition and incubation, in which an effectivequantity of an antibodies porter is added to the cellular antigen,preferably 50 to 200 μl without dilution or with a maximum dilution of1:1000 into an appropriate cell culture medium. Cellular antigenstreated with the antibody porter are incubated under an atmospherecontaining an effective CO₂ quantity for 0.25 to 2 h at a temperaturebetween 35 to 40° C.

[0119] D) A second step of washing, in which the antigen bound to theantibody porter is washed with a phosphates buffer solution at pHbetween 7.0 to 7.4. Preferably repeating 3-times and avoiding that thesurface of the cellular culture becomes dry when eliminating thephosphates buffer solution.

[0120] E) A second step of addition and incubation, in which aneffective quantity of a second antibody is added to the antigen bind tothe antibody porter. The mixture obtained is incubated under anatmosphere containing an effective CO₂ quantity for 0.25 to 2 h at atemperature between 35 to 40° C. The second antibody is selectedpreferably among antibodies from a different specie to that of theantibody porter and they can be anti-Fc of human IgG, IgA and IgM or ofthe animal in study, or antibodies anti-Fc of IgM or of IgG depending onthe nature of the monoclonal antibody when this is the antibody porter.Second antibody is used at a final dilution from 1:25 to 1:500 into anappropriate cell culture medium and it is conjugated to a substance orfluorescent substratum, preferably to FITC.

[0121] F) A third step of washing, in which the antigen bound to theantibody porter and to the second antibody is washed with a phosphatesbuffer solution at pH between 7.0 to 7.4. Preferably repeating 3-timesand avoiding that the surface of the cellular culture becomes dry wheneliminating the phosphates buffer solution.

[0122] G) An step of analysis, in which micro covers glass is mountedpreferably on a slide with a fluorescence protector such as VectaShieldto be observed in a confocal microscope, or with epifluorescence andoptics of Nomarski.

[0123] In a specific embodiment, the effective quantity of CO₂ isattained with 1 to 10% in volume with regard to air, while the effectivequantity of phosphates buffer solution is attained with 1 to 10 ml. Thefluorescent substrate can also be selected from the group consisting ofphycoerythrin, Cy3 and Percp.

[0124] In another preferred embodiment of the immunofluorescence method,microsections of an organ from humans or animals can be used as antigen,instead of a cellular culture as it was described previously. With theexception of this difference regarding the antigen, the steps of thisimmunofluorescence method are as those described in subparagraphs (B) to(G) for the immunofluorescence method in which a cellular culture isused as antigen.

[0125] Finally, the cellular-ELISA method includes the following steps:

[0126] A) A step of culture, in which an effective quantity of cellularantigen is added to each one of the wells of a microtiter plate,preferably 1×10⁵ cells, this antigen being cultivated until theconfluence in the wells reaches 100%.

[0127] B) A first step of addition and incubation, in which an effectivequantity of a blocking solution is added to each one of the wells ofmicrotiter plate, and said plate is incubated between 35 to 40° C. for0.5 to 1 h.

[0128] C) A step of elimination of blocking solution, avoiding that thesurface of the cellular culture becomes dry when eliminating theblocking solution, which can damage cellular antigens.

[0129] D) A second step of addition and incubation, in which aneffective quantity of an antibody porter is added to each one of thewells of microtiter plate in an antibody porter dilution from 1:5 to1:1000 into blocking solution; said microtiter plate being incubated for0.25 to 2.0 h at a temperature between 35 to 40° C. in presence of aneffective quantity of CO₂.

[0130] E) A first step of washing, in which cell cultures are washedwith the blocking solution, preferably repeating 3-times and avoidingthat the surface of the cellular culture becomes dry when eliminatingthe blocking solution.

[0131] F) A third step of addition and incubation, in which an effectivequantity of a second antibody is added to each one of the wells ofmicrotiter plate. This plate is incubated for 0.25 to 2 h at atemperature between 35 to 40° C. in presence of an effective quantity ofCO₂. Second antibody is selected preferably among antibodies from adifferent specie to that of the antibody porter and they can be anti-Fcof human IgG, IgA and IgM or of the animal in study, or antibodiesanti-Fc of IgM or of IgG depending on the nature of the monoclonalantibody when this is the antibody porter. Second antibody is used at afinal dilution from 1:1000 to 1:3500 into blocking solution and isconjugated to an enzyme preferably peroxidase.

[0132] G) A second step of washing, in which microtiter plate is washedwith the blocking solution, preferably repeating 3-times and avoidingthat the surface of the cellular culture becomes dry when eliminatingthe blocking solution.

[0133] H) A fourth step of addition and incubation, in which aneffective quantity of peroxidase substrates is added to each one of thewells of microtiter plate, being incubated said plate for 0.1 to 0.5 hat a temperature between 35 to 40° C., stopping the peroxidase reactionby means of an effective quantity of sulfuric acid.

[0134] I) A step of analysis, in which microtiter plate is analyzed in areading device for ELISA, preferably at 492 nm.

[0135] In a specific embodiment, the effective quantity of CO₂ isattained with 1 to 10% in volume with regard to air, while the effectivequantity of phosphates buffer solution is attained with 1 to 10 ml.

[0136] The blocking solution, includes a buffer solution at pH between7.0 to 7.4, and at least a solution with a high content of proteins,preferably fetal calf serum at 5%, volume by volume, with or without aneffective quantity of a lipidic particles inducer agent, selectedpreferably among solutions from 0.1 to 10 mM of CaCl₂, MnCl₂,chlorpromazine, procainamide or combinations of the same ones.

[0137] On the other hand in another specific modality of this method,the second antibody can be conjugated to the enzyme alkalinephosphatase, instead of peroxidase, in this case the correspondingalkaline phosphatase substrates are used.

[0138] Another aspect of the present invention, is to develop an invitro diagnosis instrument for illnesses associated withantiphospholipid antibodies, useful to carry out the method of thepresent invention. This diagnosis instrument includes at least anindicator reagent to detect the presence of lipidic particles oranti-lipidic particles antibodies in a sample of an individual havingclinical characteristics of primary antiphospholipid syndrome (Table 1),or of the illnesses associated to secondary antiphospholipid syndrome(Table 1) and who does not present yet anti-cardiolipin antibodies,lupus anticoagulant, anti-DNA or anti-nuclear antibodies; media to allowthe reaction of the sample with the indicator reagent; and, proceduresto make evident this reaction.

[0139] In a preferred embodiment, the indicative reagent is selectedamong liposomes with lipidic particles in their surface, neoplasticcells, anti-lipidic particles polyclonal antibodies, and/or anti-lipidicparticles monoclonal antibodies.

[0140] In another preferred embodiment, the sample is selected amongcells and plasma or serum of the individual. Furthermore, the medium toallow the reaction include at least a regulating solution of thereaction and at least a device to keep the reagent, the sample and theregulating solution.

[0141] The regulating solution is selected preferably among buffersolutions at pH between 7.0 to 7.4, with or without a lipidic particlesinducer agent, and phosphate buffer solutions at pH between 7.0 to 7.4,with or without a lipidic particles inducer agent.

[0142] On the other hand, the device to keep the reagent, the sample andthe regulating solution is selected preferably among tubes forcentrifugation, microtiter plates containing micro cover glasses; ELISAmicrotiter plates with a high lipidic antigens binding property; and,microtiter plates for cellular-ELISA. In the modality in which ELISAmicrotiter plates and/or cellular-ELISA microtiter plates are used, thediagnosis set also includes a blocking solution that includes a buffersolution at pH between 7.0 to 7.4, a solution with a high content ofproteins, and an effective quantity of a lipidic particles induceragent, the proteins preferably being selected between gelatin and fetalcalf serum at a concentration of 0.4 to 5%, weigh by volume, or volumeby volume, respectively.

[0143] On the other hand, the procedures to make evident the reactionare selected between fluorescent procedures and enzymatic procedures,preferably reactions of antibodies conjugated to a fluorochrome,preferably to fluorescein isothiocyanate or conjugated to an enzyme,preferably to peroxidase.

[0144] Regarding the individual sample, this is selected preferablybetween plasma or serum of the ill subject and cells from organs of theill individual.

[0145] An additional aspect of the present invention, consists ofpreventing or treating illnesses associated with antiphospholipidantibodies by means of the administration of a therapeutically effectivequantity of a drug for inhibition or blocking of the anti-lipidicparticles antibodies from sick persons, or, by means of theadministration of a therapeutically effective quantity of a stabilizerdrug to achieve the stabilization of cellular membranes from sickpersons. The above-mentioned processes are achieved in vitro by means ofinhibition or blocking of the anti-lipidic particles antibodies fromsick persons with phosphorylated haptens, which are chemical substancesthat are part of the polar region of the cellular membrane lipids; in asimilar way as it has been demonstrated in the inhibition of H308monoclonal antibody by phosphorylcholine and glycerolphosphoryl-cholinehaptens (Aguilar, op. cit. 1997).

[0146] Regarding the stabilization of cellular membranes, atherapeutically effective quantity of antimalaric drugs, which have alsobeen used in the treatment of some illnesses of the antiphospholipidsyndrome, as rheumatoid arthritis and systemic lupus erythematosus(Gibson et al., 1987, Br. J. Rheumatol. 26:279-285), is used. Amongthese drugs, it is possible to mention: chloroquine, hydroxichloroquine,amodiaquin, quinacrine or primaquine; or polyamines such as putrescine,spermidine or spermine; these polyamines are polycations which stabilizecellular membranes (Schuber, 1989, Biochem. J. 260:1-10). Both type ofdrugs avoid the formation of lipidic particles in membranal models suchas liposomes or in cellular membranes, which avoids the subsequentbinding of anti-lipidic particles antibodies, according with studiescarry out by our investigation group.

[0147] When “a therapeutically effective quantity” of a drug withinhibitory properties is used in the present invention, it means aquantity of the inhibitor drug that when is administered to a illsubject produces the blocking of anti-lipidic particles antibodiescirculating in the blood stream of the subject under treatment. “Atherapeutically effective quantity” of a stabilizer drug, is a quantityof the stabilizer drug that when it is administered to an ill subject itproduces the stabilization of cellular membranes in the individual undertreatment, so that more anti-lipidic particles antibodies are no longergenerated in this subject; or that the anti-lipidic particles antibodiespresent in the ill individual no longer react with cellular membranesbecause these membranes no longer present lipidic particles.

[0148] Studies on inhibition of anti-lipidic particles antibodies werecarried out using liposomes as antigens and the antigen-antibodyreaction was analyzed by the liposomal-ELISA method, which includes thefollowing steps:

[0149] A) A first step of addition and incubation, in which an effectivequantity of an antigen suspension is added to each one of the wells ofthe ELISA microtiter plate. This microtiter plate is selected amongthose with a high lipidic antigens binding property and said microtiterplate is incubated between 25 to 30° C. for 0.25 to 2 h.

[0150] B) A second step of addition and incubation, in which aneffective quantity of a blocking solution is added to each one of thewells of the ELISA microtiter plate with a high lipidic antigens bindingproperty, and said microtiter plate being incubated at a temperaturebetween 25 to 30° C. for 0.25 to 2 h.

[0151] C) A step of elimination of blocking solution, preferably bysuction, avoiding that microtiter plate becomes dry when blockingsolution is eliminated, because liposomal antigens can be damaged.

[0152] D) A step of inhibition of the antibody porter, in which theantibody porter is incubated with a chemical substance, or hapten, thatwill inhibit the active site that recognizes the antigen in the antibodyporter.

[0153] E) A third step of addition and incubation, in which an effectivequantity of the antibody porter inhibited by the hapten was quicklyadded, to avoid that microtiter plate becomes dry, to each one of thewells using an antibody porter dilution from 1:5 to 1:1000 into blockingsolution. This microtiter plate being incubated for 0.25 to 2 h at atemperature between 25 to 30° C.

[0154] F) A first step of washing, in which the microtiter plate iswashed with the blocking solution, preferably repeating 4-times andavoiding that microtiter plate becomes dry when eliminating the blockingsolution.

[0155] G) A fourth step of addition and incubation, in which aneffective quantity of a second antibody is added to each one of thewells of microtiter plate. This plate is incubated in the darkness for0.25 to 2 h at a temperature between 25 to 30° C. Second antibody isselected preferably among antibodies from a different specie to that ofthe antibody porter and they can be anti-Fc of human IgG, IgA and IgM orof the animal in study, or antibodies anti-Fc of IgM or of IgG dependingon the nature of the monoclonal antibody when this is the antibodyporter. Second antibody is used in a final dilution into blockingsolution between 1:000 and 1:3500 and it is conjugated to an enzymepreferably to peroxidase.

[0156] H) A second step of washing, in which the microtiter plate iswashed with the blocking solution, preferably repeating 4-times andavoiding that microtiter plate becomes dry when eliminating the blockingsolution.

[0157] I A fifth step of addition and incubation, in which an effectivequantity of the peroxidase sustrates is added to each one of the wellsand this microtiter plate is incubated for 0.1 to 0.5 h at a temperaturebetween 35 and 40° C., stopping the peroxidase reaction by adding aneffective quantity of sulfuric acid.

[0158] I) A step of analysis, in which microtiter plate is analyzedusing a reading device for ELISA plates, preferably at 492 nm.

[0159] In a specific embodiment, the antigen suspension is obtained bysuspending liposomes in a buffer solution at pH between 7.0 to 7.4, in arelationship from 1 to 5 mmole of antigen per liter of buffer solution.

[0160] The blocking solution, includes a buffer solution at pH between7.0 to 7.4, and a solution with a high content of proteins, preferablygelatin at 0.4%, weight by volume, with or without an effective quantityof a lipidic particles inducer agent, preferably with the effectivequantity and the lipidic particles inducer agent used to form theantigen.

[0161] In a preferred embodiment, the effective quantity of the antigensuspension in the stage A it is of 50 to 100 μl. The second antibody canbe also conjugated to the enzyme alkaline phosphatase, instead ofperoxidase, in this case the corresponding alkaline phosphatasesubstrates are used.

[0162] In a specific embodiment, the hapten solution is obtaineddissolving hapten in a buffer solution at pH between 7.0 and 7.4, in arelationship from 0.1 to 10 mmoles of hapten per liter of buffersolution.

[0163] In relation to the stabilization of membranes with drugs thatavoid the formation of lipidic particles in liposomal model membranes orin cellular membranes, which in turn avoid the subsequent union ofanti-lipidic particles antibodies, the studies were carried out withliposomal or cellular antigens using the cytofluorometry method. In afavorite modality this method includes the following steps:

[0164] A) A first step of incubation, in which the antigen suspension,liposomes or cells, are incubated with a drug that stabilizes itslipidic bilayers, this drug being used at a concentration of 0.1 up to100 mM, the obtained mixture is incubated for 0.25 to 2 h at atemperature between 35 and 40° C.

[0165] B) A first step of addition and incubation, in which the antibodyporter is added to the antigen stabilized with the stabilizer drug, thisantibody porter is diluted from 1:5 up to 1:1000 into a buffer solutionat pH between 7.0 to 7.4, and the resulting mixture is incubated for0.25 to 2 h at a temperature between 35 and 40° C.

[0166] C) A first step of washing, in which the antigen stabilized withstabilizer drug and bound to the antibody porter is washed with a buffersolution at pH between 7.0 to 7.4, with or without an effective quantityof a lipidic particles inducer agent, preferably with the effectivequantity of the inducer agent used to obtain the antigen.

[0167] D) A step of recovery, in which the antigen stabilized withstabilizer drug and bound to the antibody porter is recovered preferablyby centrifugation.

[0168] E) A second step of addition and incubation, in which aneffective quantity of a second antibody is added to the antigenstabilized with the stabilizer drug and bound to antibody porter. Theresulting mixture is incubated for 0.25 to 2 h in the darkness at atemperature between 35 to 40° C. Second antibody is selected preferablyamong antibodies from a different specie to that of the antibody porterand they can be anti-Fc of human IgG, IgA and IgM or of the animal instudy, or antibodies anti-Fc of IgM or of IgG depending on the nature ofthe monoclonal antibody when this is the antibody porter. Secondantibody is used at a final dilution between 1:25 to 1:500 into a buffersolution at pH between 7.0 to 7.4, and it is conjugated to a substanceor fluorescent substratum, preferably to FITC.

[0169] F) A second step of washing, in which the antigen stabilized withthe stabilizer drug and bound to the antibody porter and the secondantibody is washed with a buffer solution at pH between 7.0 to 7.4, withor without an effective quantity of a lipidic particles inducer agent,preferably with the same quantity and the same inducer agent used toobtain the antigen.

[0170] G) A step of suspension and analysis, in which the antigenstabilized with the stabilizer drug and bound to the antibody porter andthe second antibody is suspended in a transporting solution, selectedpreferably between FACS Flow (Beckton Dickinson Co.) and Haema Line 2(Serotono-Baker Diagnostics, INC) in a relationship from 1 to 5 mmole ofthe antigen in a liter of solution; this solution being preferablyfiltered previously with a 0.22 μm Millipore filter pore diameter, theobtained mixture being analyzed in a flow cytometer, preferably with asingle 488 nm argon laser beam.

[0171] In a preferred embodiment, the antigen suspension is obtainedsuspending the antigen in a buffer solution at pH between 7.0 to 7.4, ina relationship of 1 to 5 mmole per liter of buffer solution forliposomal antigen. Furthermore, fluorescent substrate can also beselected from the group consisting of phycoerythrin, Cy3 and Percp.

[0172] This antigen is incubated with a drug that stabilizes its lipidicbilayers, this drug being used at a concentration of 0.1 up to 100 mM,the resulting mixture is incubated for 0.25 to 2 h at a temperaturebetween 35 and 40° C.

[0173] In a specific embodiment the antigen can be a liposomesuspension, or cells from human or animals.

[0174] The various aspects of the present invention, will be moreclearly illustrated by the following examples, which are presented withillustrative purposes only and they should not be interpreted into alimitative form.

EXAMPLES

[0175] Liposomal antigens used in the examples were characterized bytheir ³¹P nuclear magnetic resonance spectra. These spectra showedlipids associated in bilayers or in lipidic particles in the liposomesas was previously described by Baeza et al. (op. cit., 1995), Aguilar(op. cit., 1997) and Aguilar et al., (op. cit., 1999).

Example 1 Indirect Detection by the Liposomal-ELISA Method of LipidicParticles Through the Detection of Anti-Lipidic Particles Antibodies inSera from Patients with the Antiphospholiid Syndrome

[0176] Costar microtiter plates, with 96 flat-bottom wells with a highlipidic antigens binding property (Costar Co. Cambrige, USA), werecoated by the addition of 100 μl per well of liposomes made fromegg-yolk phosphatidylcholine:phosphatidate (2:1 mole ratio) in Tris-NaClbuffer (10 mM, 1 mM) pH 7, containing 0.1 μmol of phosphatidate, andtreated with 5 mM CaCl₂ to induce lipidic particle formation. Microtiterplates were incubated 1 h at room temperature. After microtiter plateswere incubated they were blocked for 1 h at room temperature by additionof 200 μl per well of 0.4% (w/v) gelatin in Tris-NaCl buffer (10 mM, 1mM) pH 7, containing CaCl₂ 5 mM. Then the blocking solution wasdiscarded by suction and 100 μl of human sera, from patients with theantiphospholipid syndrome, at 1:50 dilution using blocking solution werequickly added to each well in duplicate, to avoid that these wellsbecomes dry; all solutions were added subsequently in the same way. As apositive control, the supernatant of a hybridoma containing a monoclonalantibody against lipidic particles, from IgM isotype, at 1:100 dilutionusing blocking solution were added to four wells. Human sera were heatedpreviously at 56° C. for 30 min for the inactivation of the complement.After microtiter plates were incubated 1 h at room temperature they werewashed 4-times with 500 μl of blocking solution. Then 100 μl ofperoxidase-conjugated goat anti-Fc of human IgG, IgA and IgM antibodiesor anti-Fc of mouse IgM antibodies at 1:2000 dilution into blockingsolution were added to each well, respectively, as second antibody.After 1 h of incubation at room temperature microtiter plates werewashed 4-times again with the blocking solution and 100 μl of freshlyprepared peroxidase substrates were added to each well (10 mgo-phenylendiamine, 25 ml Tris-NaCl buffer (10 mM, 1 mM) pH 7, and 20 μlof 30% H₂O₂) and allowed to incubate in an oven at 37° C. for 20 min.Enzyme reaction was stopped by addition of 50 μl per well of 2.5 Msulfuric acid. Absorbances were read at 492 nm in an ELISA Labsystemsreader Multiskan MS model; duplicate values were averaged for each serumsample tested.

[0177] As negative controls the second antibody was added to wells induplicate in the absence of human sera; in addition, human sera and thesecond antibody were added to wells in duplicate without liposomalantigens.

[0178] Results obtained by the liposomal-ELISA method were expressed inArbitrary Units (AU) which are determined by the following equation:${AU} = \frac{{AsP} - {AsW}}{{AsH} - {AsW}}$

[0179] Where:

[0180] AsP=Absorbance at 492 nm of patients sera;

[0181] AsW=Absorbance at 492 nm of the control without human sera; and

[0182] AsH=Absorbance at 492 nm of healthy blood donators sera.

[0183] To determine the isotype of anti-lipidic particles antibodies,human sera that gave positive reaction were analyzed again butperoxidase-conjugated goat anti-Fc of human IgG or IgM antibodies wereused as a second antibody; in order to determine whether theanti-lipidic particles antibodies correspond to the IgG or IgM isotype,respectively.

[0184] Analyzed Human Sera.

[0185] Sera studied were obtained from the Bank of the Laboratory ofImmunology of the Specialities Hospital of the Medical Center “La Raza”,from México, D. F., México, and they came from thirty patients positivefor anti-cardiolipin antibodies of the IgM or IgG isotype. Elevenpatients meet with four or more of the American Rheumatism Associationcriteria for systemic lupus erythematosus (Tan et al., 1982, ArthritisRheum. 25:1271-1277), twelve meet with the criteria for the primaryantiphospholipid syndrome (Asherson et al., op. cit., 1996; Piette etal., 1993, J. Rheumatol. 20:1802-1804), and seven for theantiphospholipid syndrome secondary to systemic lupus erythematosus(Asherson et al., op. cit., 1996) (Table 1).

[0186] Anti-cardiolipin antibodies were detected using cardiolipincoated to ELISA microtiter plates as antigen (Loizou et al., 1985, Clin.Exp. Immunol. 62:738-745). Results are also expressed in Arbitrary Units(AU) and they are considered positive when they have values≧1.9 AU forIgG isotype, and≧2.4 AU for IgM isotype (Loizou et al., op. cit., 1985).All patients' sera were positive for IgG isotype and some of them werepositive for IgM isotype (Table 2). TABLE 1 Criteria for theclassification of primary antiphospholipid syndrome, systemic lupuserythematosus, and antiphospholipid syndrome secondary to systemic lupuserythematosus from the American Rheumatism Association. Antiphospholipidsyndrome Systemic lupus Primary antiphospholipid secondary to systemiclupus erythematosus syndrome erythematosus Serositis: Venous andarterial thrombosis: Malar rash Pleuritis Renal complications Discoidrash Pericarditis Pulmonary embolism Oral or pharyngeal Cerebralischemia ulceration Necrotic skin ulcerations Frank arthritis Myocardialinfarction with uremia Nephropathy Nervous system complications:Persistent proteinuria Stroke and transient attack greater than 0.5g/day Neurological disorders Neurologic disorders: Haematologicaldisorders: Pleuritis, in the absence of Seizures Thrombocytopeniapulmonary embolism. Psychosis Haemolytic anaemia Pericarditis, in theabsence of myocardial infarction or uremia HaematologicalAntiphospholipid antibodies: Antibodies to native DNA disorders:Anti-cardiolipin Antiphospholipid Thrombocytopenia Lupus anticoagulantantibodies Haemolytic Anti- Anti-β₂-glycoprotein I anaemiaphosphatidylethanolamine antibodies Anti-phosphatidylserine ImmunologicAnti-β₂-glycoprotein I Venous and arterial disorders: antibodiesthrombosis: False positive VDRL Renal complications Antibodies toPulmonary embolism dsDNA Cerebral ischemia Antinuclear Necrotic skinulcerations antibodies Myocardial infarction with uremia Recurrent fetalloss Lymphopenia less that 1000/μl Recurrent fetal loss

[0187] TABLE 2 Detection of anti-cardiolipin and anti-lipidic particlesantibodies in human sera Anti-lipidic particles Anti-lipidic particlesAntibodies antibodies (liposomal antigen made (liposomal antigen madefrom Anti-cardiolipin from phosphatidylcholine: Anti-cardiolipinphosphatidylcholine: Antibodies phosphatidate (2:1) + CaCl₂) antibodiesphospatidate (2:1) + CaCl₂) (ELISA) (Cytofluorometry) (ELISA)(Cytofluorometry) Healthy IgM IgG Positive results at: IgM IgG Positiveresults at: blood (+) ≧ 2.4 (+) ≧ 1.9 (+) D ≧ 0.5, p < 0.001 Patients'sera (+) ≧ 2.4 (+) ≧ 1.9 (+) D ≧ 0.5, p < 0.001 donators AU AU(Polyvalent) and diagnostic AU AU Polyvalent IgM IgG  1H — — — AC11 PAPS— 7.5 D = 0.76 — D = 0.54  2H — — — AC12 PAPS — 56.3 D = 0.77 — D = 0.70 3H — — — AC13 SLE 5.24 17.2 D = 0.77 D = 0.65 D = 0.70  4H — — — AC14SLE — 10.6 D = 0.74 D = 0.65 D = 0.62  5H — — — AC15 PAPS — 6.7 D = 0.74D = 0.50 D = 0.84  6H — — — AC16 SLE — 2.52 D = 0.75 D = 0.56 D = 0.59 7H — — — AC17 SLE — 4.3 D = 0.75 — D = 0.59  8H — — — AC18 SLE + APS —67.4 D = 0.73 — D = 0.63  9H — — — AC19 SLE — 13.6 D = 0.73 D = 0.72 D =0.73 10H — — — AC20 SLE — 9.3 D = 0.75 D = 0.52 D = 0.73 11H — — — AC21PAPS — 3.36 D = 0.75 — D = 0.62 12H — — — AC22 SLE + APS 2.8 15.4 D '20.56 D = 0.52 D = 0.61 13H — — — AC23 SLE + APS 19.2 D = 0.59 — D = 0.6114H — — — AC24 PAPS — 18.0 D = 0.61 — D = 0.61 15H — — — AC25 PAPS 3.9516.3 D = 0.53 — D = 0.62 16H — — — AC26 SLE 3.06 9.2 D '2 0.53 — D =0.62 17H — — — AC27 PAPS — 8.6 D = 0.51 — D = 0.54 18H — — — AC28 PAPS —11.5 D = 0.51 — D = 0.54 19H — — — AC29 SLE — 11.08 D = 0.43 D = 0.52 D= 0.57 20H — — — AC30 PAPS — 14.7 N/D D = 0.52 D = 0.57 21H — — — AC31SLE + APS — 19.4 D = 0.66 D = 0.66 D = 0.50 22H — — — AC32 SLE + APS 3.039.6 D '2 0.56 — D = 0.57 23H — — — AC33 PAPS — 23.7 D = 0.56 — D = 0.5424H — — — AC34 SLE + APS — 34.4 D = 0.56 — D = 0.74 25H — — — AC35 PAPS4.0 18.0 D '2 0.66 D = 0.56 D = 0.75 26H — — — AC36 SLE 44.0 158.0 D =0.64 D = 0.60 D = 0.64 27H — — — AC37 SLE — 11.0 D '2 0.64 D = 0.70 D =0.75 28H — — — AC38 PAPS 3.0 2.0 D = 0.64 D = 0.59 D = 0.75 29H — — —AC39 SLE + APS — 52.0 D = 0.64 D = 0.76 D = 0.75 30H — — — AC40 SLE 4.018.0 D = 0.66 D = 0.56 D = 0.75

[0188] Sera from healthy blood donators, in other words, of healthysubjects which were used as negative controls in the analyzedimmunoreactions, did not show anti-cardiolipin antibodies from IgM orIgG isotype (Table 2). These sera came from the Bank of Blood of theMedical Center “La Raza”, from México, D. F., México.

[0189] Patients' sera and sera from healthy blood donators were suppliedus by Dr. Carlos Lavalle Montalvo, Manager of the Infectology Hospitalof the Medical Center “La Raza”, from México, D. F., México.

[0190] Results of the Detection by the Liposomal-ELISA Method ofAnti-Lipidic Particles Antibodies in Human Sera.

[0191] Reaction of human sera, from healthy blood donators or frompatients with the antiphospholipid syndrome, with liposomal antigensmade from egg-yolk phosphatidylcholine:phosphatidate (2:1 mole ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 5 mM CaCl₂ to induce lipidic particlesformation, is showed in FIG. 1. Immunoreaction of patients sera with thelipidic particles was clearly different from that of healthy blooddonators sera, or control sera, since the reaction with the peroxidasesubstrates was negative when control sera were used. In general, controlserum gave values smaller than one AU. All the values from control serawere combined to obtain the arithmetic mean and the standard deviation.We then consider as positive all results greater than 3 standarddeviations from the mean. After this analysis, sera from the 30 healthyblood donors were mixed and the mixture was used as a control sera forsubsequent analysis. In FIG. 1 the dark line indicates the upper limitabove which the reactions of sera with lipidic antigens are positive.The reaction of most patients sera was clearly positive, with values ofAU higher than 6.

[0192] Arbitrary Units of 7 sera (AC12, AC14, AC15, AC16, AC31, AC32 andAC34) are showed in FIG. 1. These sera are representative of the 30analyzed sera. AC12 and AC15 sera correspond to patients with primaryantiphospholipid syndrome (PAPS); AC14 and AC16 sera from patients withsystemic lupus erythematosus (SLE) and AC31, AC32, and AC34 sera frompatients with antiphospholipid syndrome secondary to systemic lupuserythematosus (APS+SLE). In this Figure as a positive control it isshowed the reaction of H308 monoclonal antibody with liposomal antigensfrom egg-yolk phosphatidylcholine: phosphatidate (2:1 mole ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 5 mM CaCl₂ to induce lipidic particlesformation. Peroxidase-conjugated goat anti-Fc of human IgG antibodieswere used as second antibody for the human sera and anti-Fc of mouse IgMfor monoclonal antibody, both at 1:2000 final dilution.

[0193] An important particularity of liposomal-ELISA method consists inthat it allows the simultaneous determination of anti-lipidic particlesantibodies in at least 40 sera samples by microtiter plate, each one induplicate; for this reason this method can be easily applied to thediagnosis of illnesses where this type of antibodies is presented.

Example 1A Comparative Study when Antigens Without Lipidic Particles andSera from Patients with the Antiphospholipid Syndrome are Used in theLiposomal-ELISA Method

[0194] Example 1 was repeated but using as antigens “rigid” liposomesmade from dipalmitoylphosphatidylcholine:egg-yolkphosphatidylcholine:dipalmitoylphosphatidate (1.2:0.8:1.0 mole ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, which when incubated with 5 mMBaCl₂ they conserve the smooth surface of bilayer. In this case, thereaction of the substrates of peroxidase-conjugated to the secondantibody was negative. Because liposomes did not have any lipidicparticles, therefore the anti-lipidic particles antibodies did not reactwith them and consequently the second antibody did not bind theseliposomes, which explains the negative reaction of peroxidasesubstrates. These results discard the possibility that anti-lipidicparticles antibodies recognize a lipid-divalent cation complex and/orthe reduction in the liposomal surface charge due to the binding ofdivalent cations without affecting the bilayer lipid arrangements andtheir change to lipidic particles arrangements (Aguilar et al., op.cit., 1999).

Example 1B Comparative Study when Antibodies Different to theAnti-Lipidic Particles Antibodies and Liposomal Antigens Bearing LipidicParticles are Used in the Liposomal-ELISA Method

[0195] Example 1 was repeated with some modifications. In thisexperiment liposomes made from egg yolkphosphatidylcholine:phosphatidate (2:1 mole ratio) in Tris-NaCl buffer(10 mM, 1 mM) pH 7, containing 0.1 μmol of phosphatidate, and treatedwith 5 mM CaCl₂ to induce lipidic particles formation, were incubateddirectly with peroxidase-conjugated goat anti-Fc of human IgG, IgA andIgM antibodies, or with the supernatant of a hybridoma producingunrelated monoclonal antibodies, as those against a membranal protein ofTrichinella spiralis, from IgM isotype, and peroxidase-conjugated goatanti-Fc of mouse IgM antibodies. In both cases, the reaction withperoxidase substrates was negative, because in absence of humananti-lipidic particles antibodies or mouse anti-lipidic particlesmonoclonal antibody the second antibody, peroxidase-conjugated goatanti-Fc of human IgG, IgA and IgM or anti-Fc of mouse IgM antibodies, donot bind directly to lipidic particles induced by calcium in liposomalantigens.

Example 1C Comparative study when Anti-Lipidic Particles Antibodies andSecond Antibody are Used in Absence of Liposomal Antigens BearingLipidic Particles in the Liposomal-ELISA Method

[0196] Example 1 was repeated but in the absence of liposomal antigens,in consequence the reaction of substrates of the peroxidase-conjugatedto second antibody was negative. Due to anti-lipidic particlesantibodies do not bind directly to microtiter plate which could give afalse positive result, because microtiter plate was blocked with thegelatin that is used in this methodology, consequently the secondantibody do not bind to microtiter plate which explains the negativereaction of the substrates of peroxidase-conjugated to the secondantibody.

[0197] From these examples we can conclude that, in a preferredembodiment of the present invention, a diagnosis kit particularly usefulfor the detection by the protocol of liposomal-ELISA of anti-lipidicparticles antibodies in at least a serum sample from a subject sufferingfrom an illnesses related with antiphospholipid antibodies include: anindicator reagent including, firstly, at least liposomes with lipidicparticles and, secondly, at least an anti-lipidic particles monoclonalantibody; at least a blocking solution to prevent possible falsepositive results from occurring; at least a buffer solution, as a mediumto allow the reaction between the sample coming from the sick personwith this indicator reagent to proceed; enzymatic media that includepreferably the peroxidase enzyme, to make evident this reaction; and atleast a sample of a reference serum coming from a healthy individual, asnegative control of the reaction with liposomal antigens bearing lipidicparticles.

[0198] In this preferred embodiment of the diagnosis kit, the serumsample coming from the ill subject is made react with the indicatorreagent containing liposomes bearing lipidic particles. Also, theindicator reagent containing liposomes with lipidic particles is madereact with the anti-lipidic particles monoclonal antibody, as a positivecontrol, showing that the system to detect the reaction betweenanti-lipidic particles antibodies from the serum of a subject sufferingfrom an illnesses associated with antiphospholipid antibodies and theantigen bearing lipidic particles works correctly.

[0199] In an alternative embodiment, the diagnosis kit also comprises assome part of it one or more microtiter plate(s) as recipient(s) for thedevelopment of the reaction. In the same fashion, in another alternativemodality the sample of a healthy individual serum can not be included inthe same kit, being obtained, in this case, from an external source.This serum sample is coming from a healthy individual who does notpresent an illness associated with antiphospholipid antibodies.

Example 2 Detection by Cytofluorometry of the Liposomal AntigensAutofluorescence

[0200] Samples of 100 μl of liposomes made from egg yolkphosphatidylcholine: phosphatidate (2:1 mole ratio) in Tris-NaCl buffer(10 mM, 1 mM) pH 7, containing 0.1 μmol of phosphatidate were analyzedin a FACSCalibur Flow Cytometer equipped with a single 488 nm argonlaser beam (Beckton Dickinson). Autofluorescence readings were obtainedfrom 10,000 liposomes in a logarithmic mode and they were made in theFL-1 channel at 748 V (Baeza et al., op. cit. 1995). The obtained datawere analyzed with the Cellquest program (Beckton Dickinson).

[0201] Autofluorescence histograms obtained from egg yolkphosphatidylcholine: phosphatidate (2:1 mole ratio) liposomes showedvalues between 1 to 10 fluorescence units (a, FIG. 2A). The detection ofliposomal autofluorescence allowed the application of cytofluorometry tothe analysis of immunologic reactions where liposomal antigens are used.Liposomal autofluorescence (a, FIG. 2A) was not modified when liposomeswere incubated with 5 mM CaCl₂ (c, FIG. 2B), which indicates that thepresence of lipidic particles in liposomes did not modify the liposomalautofluorescence. Furthermore, this fluorescence was not modified by theaddition of FITC-conjugated goat anti-Fc of human IgG, IgA and IgM oranti-Fc of mouse IgM antibodies as second antibodies, which indicatesthat these antibodies do not bind directly liposomal antigens, andtherefore they can not produce a false positive reaction. Results withthe FITC-conjugated goat anti-Fc of human IgG, IgA and IgM antibodies assecond antibody at 1:200 final dilution are shown in: b, FIG. 2A and d,FIG. 2B, with liposomal antigens in absence of calcium (b, FIG. 2A) asin presence of this divalent cation (d, FIG. 2B).

[0202] Similar results were obtained with liposomes made fromphosphatidylcholine; phosphatidylcholine:cardiolipin (2:1 mole ratio);phosphatidylcholine:phosphatidylserine (4:1 mole ratio) or fromdipalmitoylphosphatidylcholine:egg-yolkphosphatidylcholine:dipalmitoylphosphatidate (1.2:0.8:1 mole ratio),respectively. Therefore the cytofluorometry can be applied in general tothe analysis of immunologic reactions where liposomal antigens withdifferent lipidic formulations are used.

Example 2A Detection by the Liposomal Cytofluorometry Method of LipidicParticles in Liposomes Using H308 Monoclonal Antibody

[0203] Samples of 100 μl of liposomes made from egg yolkphosphatidylcholine: phosphatidate (2:1 mole ratio) in Tris-NaCl buffer(10 mM, 1 mM) pH 7, containing 0.1 μmol of phosphatidate, and treatedwith 5 mM CaCl₂ to induce lipidic particles formation, were placed in14×95 mm ultracentrifuge tubes (Beckman ultra-clear No. 344060). To eachone of these aliquots the supernatant of a hybridoma, the H308, thatgenerates an anti-lipidic particles monoclonal antibody at 1:100 finaldilution into Tris-NaCl buffer (10 mM, 1 mM) pH 7, was added. Afterincubation for 1 h at 37° C., liposomes were washed with 12 ml ofTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 5 mM CaCl₂. Liposomeswere recovered by centrifugation in the ultracentrifuge Beckman L8-M, at202,000×g for 50 min at 18° C. Next FITC-conjugated goat anti-Fc ofmouse IgM antibodies at 1:200 final dilution into Tris-NaCl buffer (10mM, 1 mM) pH 7, was added to each tube as second antibody and wasincubated 1 h at 37° C. in the darkness. At the end of the incubationliposomes were washed as it was previously indicated. Finally, liposomespreparation were resuspended in 500 μl of FACS flow solution (BecktonDickinson Co.) filtered with a 0.22 μm Millipore filter pore diameter.This liposomal suspension was analyzed by cytofluorometry in aFACSCalibur Flow Cytometer equipped with a single 488 nm argon laserbeam (Beckton Dickinson).

[0204] Fluorescence readings were made in the FL-1 channel. The relativesize and/or liposomal aggregation were analyzed by diffraction of thelaser beam in the FSC (forward scatter light) channel and thegranularity or liposomal bilayers complexity was analyzed by refractionand reflection of the laser in the SSC (side scatter light) channel.Analysis of 10,000 liposomes was made in a logarithmic scale with thefollowing detectors: FSC in E00, with a detector compensation thresholdof 52 V; SSC of 401 V and FL-1 of 748 V (Baeza et al., op. cit., 1995).The obtained data were analyzed with the Cellquest program (BecktonDickinson).

[0205] “Rigid” liposomes made fromdipalmitoylphosphatidylcholine:egg-yolkphosphatidylcholine:dipalmitoylphosphatidate (1.2:0.8:1.0 mole ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, alone or incubated with BaCl₂ 5 mMwere also used as antigens.

[0206] H308 monoclonal antibody do not show any reactivity with bilayerlipid arrangements, since the fluorescence detected from smoothliposomes incubated with this monoclonal antibody (a, FIG. 3A) wassimilar to the autofluorescence of control liposomes, in Tris-NaCl ortreated with calcium, that were not incubated with monoclonal antibody(g, h, FIG. 3D). Furthermore, SSC and FSC values indicated the absenceof lipidic particles and of liposomal aggregation in smooth liposomesthat were not treated with calcium (i, FIG. 3E; k, FIG. 3F) no matter ifthey were incubated with H308 monoclonal antibody (c, FIG. 3B; e, FIG.3C). On the contrary, the 60-fold increase in the fluorescence ofliposomes treated with calcium (b, FIG. 3A) with regard to thefluorescence of liposomes with lipids in bilayers (a, FIG. 3A), with avalue in the fluorescence difference among these liposomal populationsin a logarithmic scale (D)=0.9 at p<0.001, showed the reaction of H308monoclonal antibody with the lipidic particles induced by calcium.Values of D≧0.5 at p<0.001 indicate a difference among the studiedpopulations that is highly significant from the statistical point ofview (Lampariello, 2000, Cytometry 39:179-188). Therefore, values ofD≧0.5 at p<0.001 were considered as positive results and indicative ofthe presence of anti-lipidic particles antibodies in the analyzedsamples.

[0207] On the other hand, SSC values indicated that the pattern oflipidic particles was different after the immunoreaction (d, FIG. 3B)compared with the pattern of these lipidic structures in liposomes thatwere not incubated with H308 monoclonal antibody (j, FIG. 3E); theseprofiles reflect the dynamic properties of lipidic particles. Besides,liposomal aggregation, was discarded, because FSC values that showliposomal aggregation, were similar after the immunoreaction (f, FIG.3C) to those of liposomes with lipidic particles that were not incubatedwith H308 monoclonal antibody (l, FIG. 3F).

[0208] Monoclonal antibody reaction with lipidic particles of liposomalantigens is considered as a positive reference of the reaction ofpatients' antibodies with this type of lipidic structures. Inconsequence it is necessary to include this determination as a positivecontrol in the analysis of the detection of anti-lipidic particlesantibodies in sera from human individuals or animals by liposomalcytofluorometry.

[0209] On the other hand, a monoclonal antibody, from isotype IgM,unrelated with the liposomal system analyzed, as the one directedagainst a membranal protein of Trichinella spiralis, did not show theindicate reactions for H308 monoclonal antibody with lipidic particles.Since cytofluorometry graphs obtained with this unrelated monoclonalantibody were similar to those of control liposomes treated with calciumin absence of H308 monoclonal antibody (h, FIG. 3D; j, FIG. 3E; and l,FIG. 3F).

[0210] “Rigid” liposomes made fromdipalmitoylphosphatidylcholine:egg-yolkphosphatidylcholine:dipalmitoylphosphatidate (1.2:0.8:1.0 mole ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, that incubated with 5 mM BaCl₂conserved the smooth surface of bilayers did not show any reaction withH308 monoclonal antibody, because the cytofluorometry graphs obtained(n, FIG. 3G; p, FIG. 3H; and r, FIG. 3I) were similar to those ofliposomes that were not treated with BaCl₂ neither with the monoclonalantibody (m, FIG. 3G; o, FIG. 3H; and q, FIG. 3I).

Example 2B Indirect Detection by the Liposomal Cytofluorometry Method ofLipidic Particles Through the Detection of Anti-Lipidic ParticlesAntibodies in Sera from Patients with the Antiphospholipid Syndrome

[0211] This detection is similar to the one described in Example 2A,however sera from patients with antiphospholipid syndrome were used asthe antibody carrier instead of H308 monoclonal antibody. Samples of 100μl of liposomes made from egg yolk phosphatidylcholine:phosphatidate(2:1 mole ratio) in Tris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1μmol of phosphatidate, and treated with 5 mM CaCl₂ to induce lipidicparticles formation were placed in 14×95 mm ultracentrifuge tubes(Beckman ultra-clear No. 344060). Sera from patients with theantiphospholipid syndrome at 1:50 final dilution into Tris-NaCl buffer(10 mM, 1 mM) pH 7, were added to each one of ultracentrifuge tubes andthey were incubated for 1 h at 37° C. Sera were previously heated at 56°C. for 30 min for the inactivation of the complement. After incubation,liposomes were washed with 12 ml of Tris-NaCl buffer (10 mM, 1 mM) pH 7,containing 5 mM CaCl₂. Liposomes were recovered by centrifugation in theultracentrifuge Beckman L8-M, at 202,000×g for 50 min at 18° C. Next itwas added to each tube FITC-conjugated goat anti-Fc of human IgG, IgAand IgM antibodies at 1:200 final dilution into Tris-NaCl buffer (10 mM,1 mM) pH 7, as second antibody and was incubated 1 h at 37° C. in thedarkness. At the end of incubation liposomes were washed as it waspreviously indicated. Finally, liposome preparations were resuspended in500 μl of FACS flow solution (Beckton Dickinson Co.) filtered with a0.22 μm Millipore filter pore diameter. This liposomal suspension wasanalyzed by cytofluorometry in a FACSCalibur Flow Cytometer equippedwith a single 488 nm argon laser beam (Beckton Dickinson).

[0212] Fluorescence readings were made in the FL-1 channel. The relativesize and/or liposomal aggregation were analyzed in the FSC channel andthe granularity or liposomal bilayers complexity was analyzed in the SSCchannel. Analysis of 10,000 liposomes was made in a logarithmic modewith the following detectors: FSC in E00, with a detector compensationthreshold of 52 V; SSC of 401 V and FL-1 of 748 V (Baeza et al., op.cit., 1995). The obtained data were analyzed with the Cellquest program(Beckton Dickinson).

[0213] As a negative control, the reaction of healthy blood donatorssera with liposomes made from egg yolk phosphatidylcholine:phosphatidate(2:1 mole ratio) in Tris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1μmol of phosphatidate, and treated with 5 mM CaCl₂ to induce lipidicparticles formation was analyzed. FITC-conjugated goat anti-Fc of humanIgG, IgA and IgM antibodies were used as second antibody.

[0214] Thirty healthy blood donors' sera were studied. These sera didnot present any immunoreaction with lipidic particles, sincefluorescence graphs obtained with liposomes incubated with them weresimilar to those of control liposomes that were exclusively incubatedwith 5 mM CaCl₂ (h, FIG. 3D; and j, FIG. 3E from Example 2A). After thisanalysis, sera from the 30 healthy blood donators were mixed and themixture was used as a control sera for subsequent analysis.Cytofluorometry graphs of mixed sera are presented in: a, FIGS. 4A, D,G, J, M, P, S and V; in c, FIGS. 4B, E, H, K, N, Q, T and W, and in e,FIGS. 4C, F, I, L, 0, R, U and X. FSC values (e, FIGS. 4C, F, I, L, O,R, U and X) showed the absence of liposomal aggregation by the treatmentwith healthy blood donators sera, because they were very similar tothose of liposomes control in absence of human sera indicated in: l,FIG. 3F, from Example 2A.

[0215] Immunoreaction of all patients' sera with liposomal antigenstreated with calcium showed a fluorescence 20 to 40-fold higher thanthat of control sera reaction, with a difference between liposomalfluorescence in a logarithmic scale (D)≧0.5 at p<0.001 (Table 2). Valuesof D≧0.5 at p<0.001 were considered as positive results and indicativeof the presence of anti-lipidic particle antibodies in sera analyzed, ina similar way as it was described for H308 monoclonal antibodies. Asexample, fluorescence histograms of eight sera from patients withsystemic lupus erythematosus (SLE) (AC19 and AC20), with primaryantiphospholipid syndrome (PAPS) (AC15, AC21 and AC30) or withantiphospholipid syndrome secondary to systemic lupus erythematosus(SLE+APS) (AC18, AC22 and AC31) are shown in: b, FIG. 4A; g, FIG. 4D; j,FIG. 4G; m, FIG. 4J; o, FIG. 4M; r, FIG. 4P; u, FIG. 4S; and x, FIG. 4V.In the eight sera the reaction between anti-lipidic particle antibodiescontained in patients' sera and lipidic particles of liposomal antigensalthough positive, were clearly different from each other and withregard to the reaction of H308 monoclonal antibody (compare d, FIG. 4B;h, FIG. 4E; k, FIG. 4H; n, FIG. 4K; p, FIG. 4N; s, FIG. 4Q; v, FIG. 4T;and y, FIG. 4W with d, FIG. 3B, from Example 2A), which can beattributed to the polyclonal origin of human antibodies.

[0216] SSC values (d, FIG. 4B; h, FIG. 4E; k, FIG. 4H; n, FIG. 4K; p,FIG. 4N; s, FIG. 4Q; v, FIG. 4T; and y, FIG. 4W), parameter whereliposomal bilayer complexity and therefore the presence of lipidsassociated in lipidic particles is analyzed, were similar to those ofcontrol liposomes incubated with calcium to induce lipidic particlesformation (j, FIG. 3E, from Example 2A). Therefore, SSC values showedthe presence of lipidic particles in liposomes which gave the reactionwith the anti-lipidic particles antibodies contained in patients sera.

[0217] Furthermore, the reaction of patients sera with lipidic particlesdid not show any liposomal aggregation, that could increase in anunspecific form the fluorescence registered and to give a positive falseresult, since FSC values (f, FIG. 4C; i, FIG. 4F; l, FIG. 4I; {overscore(n)}, FIG. 4L; q, FIG. 4O; t, FIG. 4R; w, FIG. 4U; and z, FIG. 4X) weresimilar after the immunoreaction to those of liposomes incubated withhealthy blood donators sera (e, FIGS. 4C, F, I, L, O, R, U and X) andwith those incubated with calcium in absence of antibodies (l, FIG. 3F,from Example 2A).

[0218] Since liposomal cytofluorometry method has a sensibility 10-foldhigher than liposomal-ELISA method in the detection of anti-lipidicparticles antibodies it must be applied when some doubtful result hasbeen obtained with liposomal-ELISA method. For example, sera such asAC27 that show by liposomal-ELISA method a value of AU<1.0, which isnegative for the detection of anti-lipidic particles antibodies, byliposomal cytofluorometry it shows a result with a value of D=0.51, atp<0.001, which is clearly positive of the presence of anti-lipidicparticles antibodies.

Example 2C Comparative Study when Liposomal Antigens Without LipidicParticles and Sera from Patients with the Antiphospholipid Syndrome areUsed in the Cytofluorometry Method

[0219] “Rigid” liposomes made fromdipalmitoylphosphatidylcholine:egg-yolkphosphatidylcholine:dipalmitoylphosphatidate (1.2:0.8:1.0 mole ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, that incubated with BaCl₂ 5 mMconserved the smooth surface, were used as antigens. In “rigid”liposomes there were not the formation of lipidic particles becausetheir rigid bilayers do not allow the lipidic movement that is requiredto form lipidic particles. These liposomes were incubated with sera frompatients with the antiphospholipid syndrome and FITC-conjugated goatanti-Fc of human IgG, IgA and IgM antibodies were used at a finaldilution of 1:200 as second antibody.

[0220] Cytofluorometry graphs obtained showed that patients sera did notpresent any reaction with “rigid” liposomes treated with BaCl₂ (b, FIG.5A; d, FIG. 5B; and f, FIG. 5C) because they were similar to those ofliposomes that were not treated with BaCl₂ (a, FIG. 5A; c, FIG. 5B; ande, FIG. 5C). Graphs showed in FIGS. 5A-5C, correspond to the reaction ofAC15 serum from a patient with primary antiphospholipid syndrome (PAPS)and it is representative of reaction of sera from the remaining patientsindicated in Table 2.

[0221] From the above mentioned examples, we can conclude that inanother favorite modality of the present invention, a diagnosis kitparticularly useful for the detection by the liposomal cytofluorometrymethod of anti-lipidic particles antibodies in sera from subjects withillnesses associated with antiphospholipid antibodies comprises: anindicator reagent, including at least liposomes bearing lipidicparticles; at least a buffer solution as medium to allow the reactionbetween the ill subject sample with this indicator reagent to proceed;and fluorescent media to make evident this reaction.

[0222] In this preferred embodiment of the diagnosis kit, sera samplecoming from ill subjects is made react with the indicator reagentcontaining liposomes bearing lipidic particles.

[0223] In an alternative embodiment, the diagnosis kit includes as somepart of it one or more tube(s) for centrifugation as recipient(s) forthe development of the reaction.

[0224] In the same fashion, in another alternative embodiment thisdiagnosis kit can include at least an anti-lipidic particles monoclonalantibody as a positive control of the antibodies reaction with liposomalantigens bearing lipidic particles, and at least a sample of a referenceserum coming from a healthy individual, as a negative control of thereaction with liposomal antigens bearing lipidic particles.

[0225] In another alternative embodiment the serum sample of a healthyindividual can be obtained from an external source. This serum sample iscoming from a healthy individual that does not present an illnessassociated with antiphospholipid antibodies.

Example 3 Direct Detection by the Immunofluorescence Method of LipidicParticles in Cells from a Subject Using H308 Monoclonal Antibody

[0226] C5337 cancer pancreas cells were used as antigens. In a cellularculture plate, with 24-wells containing sterile micro cover glasses ineach one of the wells, 1×10⁶ cells were added by micro cover glass andplate was incubated at 37° C. in an atmosphere containing 5% CO₂. When90% of cellular confluence was obtained, cells were washed twice with 2ml of incomplete DMEM cell culture medium and once with 2 ml of sterilephosphates buffer at pH 7.4. All solutions were quickly added to avoidthat cells surface becomes dry. Next, 200 μl of supernatant H308hybridoma, containing anti-lipidic particles H308 monoclonal antibody,at 1:10 dilution using incomplete DMEM cell culture medium were added,and cells were incubated for 1 h at 37° C. in presence of 5% CO₂. Afterincubation, cell cultures were washed 3-times with 2 ml of phosphatesbuffer, pH 7.4 and 200 μl of FITC-conjugated goat anti-Fc of mouse IgMantibodies at 1:200 dilution, using incomplete DMEM cell culture medium,were added. After incubation for 1 h at 37° C. in presence of 5% CO₂cell cultures were washed 3-times again with 2 ml of phosphates buffer.Finally, micro cover glasses were mounted in slides with VectaShield,these preparations were sealed, observed, and photographed withepifluorescence and optics of Nomarski using a Nikon Optiphot-2microscope.

[0227] C5337 cancer pancreas cells showed areas with a strongfluorescence intensity located in small points, in occasions abovecellular nucleus (1, FIG. 6); in some cases fluorescence was located incell junctions (2, FIG. 6). In other cases, neoplastic cells were markedin the whole surface, these cells showed a round morphology (3, FIG. 6)as that corresponding to cells that do not adhere to cell culture plateswhich can be in apoptosis, or programmed cellular death; furthermore,these cells also can not be adhered because they will be in a cellulardivision process. Immunostaining shows the reaction of monoclonalantibody with lipidic particles present in membranes of C5377 cancerpancreas cells. H308 monoclonal antibody was adsorbed with egg-yolkphosphatidylcholine:phosphatidate liposomes (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate and treated with CaCl₂ 5 mM to induce lipidic particlesformation, to confirm that the observed immunostaining was really withcellular lipidic particles. After this adsorption the supernatant ofH308 hybridoma no longer showed reaction with C5337 cells because theanti-lipidic particles antibodies from this supernatant were eliminated.

[0228] As a negative control, neoplastic cells were incubated withFITC-conjugated second antibody or with an unrelated monoclonalantibody, from IgM isotype, directed against a membranal protein ofTrichinella spiralis. In both cases there was not any reaction ofantibodies with neoplastic cells since fluorescence that showed theimmunoreaction was not observed.

[0229] These studies show that H308 monoclonal antibody besides reactingwith lipidic particles of liposomal membranes, also reacts withmembranal lipidic particles from cells in cultures. These cellsrepresent a natural antigen contrary to liposomes that are anexperimental model of cellular membranes. When tissue sections (6μm-thickness) have also been used, as human placenta sections, H308monoclonal antibody reacted with cellular membranes of this organ.Immunoreaction was different along the 9 months of life of this organand a higher quantity of lipidic particles was detected in the finalstages of pregnancy. This reaction showed that H308 monoclonal antibodyalso reacts with lipidic particles present in a natural way in thishuman organ.

[0230] In accordance with the above-mentioned studies, detection oflipidic particles in cell membranes can be used to characterize thedistinct functional states that cells go through during the differentstages of cellular cycle, including the apoptosis process, or programmedcellular death.

Example 3A Direct Detection by the Cytofluorometry Method of LipidicParticles in Cells from a Subject Using H308 Monoclonal Antibody

[0231] A sample of 100,000 plaquettes in 100 ml of Tris-NaCl buffer (10mM, 135 mM) pH 7, containing glucose 11 mM, was placed in polystyrene8×75 mm tubes. To each one of these aliquots H308 hybridoma supernatantat 1:100 dilution into Tris-NaCl buffer (10 mM, 135 mM) pH 7, containingglucose 11 mM, was added. This hybridoma generates an anti-lipidicparticles monoclonal antibody from de IgM isotype. Additionally, 5 μMadenosin diphosphate (ADP) was added to each one of the tubes and theywere incubated for 30 min at 37° C. After incubation, plaquettes werewashed with 4 ml of Tris-NaCl buffer (10 mM, 135 mM) pH 7, containingglucose 11 mM. Plaquettes were recovered by centrifugation at 200×g for5 min. After centrifugation, FITC-conjugated goat anti-Fc of mouse IgMantibodies at 1:200 final dilution, into Tris-NaCl buffer (10 mM, 135mM) pH 7, containing glucose 11 mM, were added to each tube as secondantibody and tubes were incubated for 1 h at 37° C. in the darkness. Atthe end of the incubation plaquettes were washed as it was indicatedpreviously. Finally, plaquettes were resuspended in 500 μl of FACS Flowsolution (Beckton Dickinson Co.) filtered with a 0.22 μm Milliporefilter diameter pore.

[0232] Plaquettes suspension was analyzed by cytofluorometry in aFACScalibur Flow Cytometer equipped with a single 488 nm laser beam(Beckton Dickinson).

[0233] Fluorescence readings were made in the FL-1 channel. Plaquettesrelative size and/or plaquettes aggregation were analyzed by diffractionof the laser beam in the FSC channel. Granularity or membranal plaquettecomplexity was analyzed by refraction and reflection of the laser in theSSC channel. Analysis of 10,000 plaquettes was made with the followingdetectors: FSC in E00, in a lineal mode with an amplifier gain of 5 Vand with a detector compensation threshold of 52 V; SSC of 450 V andFL-1 of 700 V, both in logarithmic mode (Baeza et al., op. cit., 1995).The obtained data were analyzed with the Cellquest program (BecktonDickinson).

[0234] Plaquettes treated as described above but without any ADPactivation were used as a negative control of the immunoreaction of themwith H308 monoclonal antibody.

[0235] Anti-lipidic particles H308 monoclonal antibody showed reactivitywith ADP activated plaquettes. Fluorescence histograms of theimmunoreaction of plaquettes without any activation or ADP activatedwere similar to those presented in: a,m, FIG. 4J, Example 2B, for AC31patient serum and control sera, respectively. Cytofluorometrichistograms showed a 10-fold fluorescence increase when ADP activatedplaquettes were used as antigens, with D=0.50 at p<0.001. In addition,graphs corresponding to values of membranal activated plaquettescomplexity and of activated plaquettes aggregation were as those showedin: n, FIG. 4K; and {overscore (n)}, FIG. 4L for the indicate sera.These results showed the higher complexity in membranal plaquettesduring their ADP activation as well as the lack of plaquettesaggregation during this process.

[0236] These results show clearly the presence of lipidic particles inplaquettes which are cellular fragments containing a residual membranewhich allows to study the structural and functional characteristics ofthis cellular organelle.

[0237] The methodology of this Example can also be used to detectlipidic particles in isolated cells, such as erythrocytes and leukocyteswhich are in different physiologic states. These studies will allow tocharacterize the physiologic states of cells by the quantity of lipidicparticles that present in their cellular membranes. This knowledge cancontribute to maintain cells in a more appropriate functional state andtherefore it can contribute to the prevention of illnesses.

Example 3B Direct Detection by the Cellular-ELISA Method of LipidicParticles in Cells from a Subject Using Anti-Lipidic ParticlesAntibodies from Sera of Patients With the Antiphospholipid Syndrome

[0238] C5337 pancreas cancer cells were used as antigens, then 1×10⁵cells was seeded in each well of a flat-bottom 96-wells microtiterplates, and they were incubated at 37° C. in an atmosphere containing 5%CO, until cell confluence in the wells reached 100%. After incubation,200 μl of a blocking solution containing Tris-NaCl buffer (10 mM, 135mM) pH 7, and 5% fetal calf serum, were added to each one of the wellsand microtiter plates were incubated for 30 min at 37° C. Additionally,the blocking solution was eliminated and 100 μl of sera from patientswith the antiphospholipids syndrome, or from healthy blood donators at1:50 final dilution, using blocking solution, were quickly added toavoid that cells surface becomes dry. All solutions were addedsubsequently in the same way. After cell cultures were incubated for 30min at 37° C. in an atmosphere containing 5% CO₂, they were washed3-times with 200 μl of blocking solution for 5 min in each washing.Next, 100 μl of peroxidase-conjugated goat anti-Fc of human IgG, IgA andIgM antibodies at 1:2000 dilution, into blocking solution, were added assecond antibody. Microtiter plates were incubated for 30 min at 37° C.in an atmosphere containing 5% CO₂. After incubation, microtiter plateswere washed as it was indicated and 100 μl of peroxidase substrates wereadded to each one of the wells and plates were again incubated for 20min at 37° C. Finally 50 μl of 2.5 M sulfuric acid were added to stopthe peroxidase reaction and the absorbency was read at 492 nm in anELISA Labsystems reader Multiskan MS model.

[0239] Sera from the thirty patients studied in Examples 1 and 2Bpresented reaction with the neoplastic cells. Arbitrary units higherthan 1 were obtained from the 492 nm absorbance readings. To confirmthat this immunoreaction was with lipidic particles present in membranesof C5337 pancreas cancer cells, patients sera were adsorbed withegg-yolk phosphatidylcholine:phosphatidate liposomes (2:1 molar ratio)in Tris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with CaCl₂ 5 mM to induce lipidic particlesformation. After this adsorption patients sera no longer showed reactionwith C5337 pancreas cancer cells because the anti-lipidic particlesantibodies were eliminated of them.

[0240] Results obtained with some patients sera are showed in FIG. 7.These sera are: AC15, AC30 and AC33 from patients' with primaryantiphospholipid syndrome; AC19 and AC29 from patients with systemiclupus erythematosus, and AC34 from a patient with antiphospholipidsyndrome secondary to systemic lupus erythematosus. Bar graphs of thedirect reaction of patients sera with C5337 cells as well as of thereaction of patients sera after their adsorption with liposomes bearinglipidic particles are showed in FIG. 7. It can be seen that the reactionof sera with cellular antigens was eliminated after their adsorptionwith liposomes bearing lipidic particles because the anti-lipidicparticles antibodies that they contained were eliminated. C5337 cellswere incubated with a serum from a healthy blood donator (FIG. 7) as anegative control of the reaction of human sera with cellular lipidicparticles. In FIG. 7 the dark line indicates the uper limit above ofwhich the reactions of sera with cellular antigens are positive.

[0241] These experiments are very important because they show that theanti-lipidic particles antibodies of sera from ill subjects, which wereprimary detected with experimental membrane models such as liposomes,also showed reaction with the lipidic particles of cellular antigens,which really represent a natural antigen as those found in humans andanimals.

Example 3C Direct Detection by the Immunofluorescence Method of LipidicParticles in Cells from a Subject Using Anti-Lipidic ParticlesAntibodies from Sera of Patients With the Antiphospholipid Syndrome

[0242] This detection was carried out as it was indicated in the Example3, with the difference that C5337 pancreas cancer cells were incubatedwith sera from patients with the antiphospholipid syndrome bearinganti-lipidic particles antibodies instead of H308 monoclonal antibody.Patients' sera were used at 1:50 dilution and FITC-conjugated goatanti-Fc of human IgG, IgA and IgM antibodies were used as secondantibody.

[0243] Neoplastic cell cultures were marked with the anti-lipidicparticles antibodies from patients' sera in a similar way as it wasdescribed for C5337 pancreas cancer cells in FIG. 6, from Example 3,showing the binding of lipidic particles from neoplastic membranes withthese anti-lipidic particles antibodies.

[0244] On the other hand, the methodology described in this Example canbe applied in an alternative way to the detection of anti-lipidicparticles antibodies in patients' sera when these antibodies have beennot yet detected by the procedures indicated in the Examples 1 and 2B.

[0245] From previous examples, we can conclude that in another favoritemodality of the present invention, a diagnosis kit particularly usefulfor the direct detection of lipidic particles in cellular antigensincludes: at least an indicator reagent including at least ananti-lipidic particles monoclonal antibody; at least a buffer solutionas a medium to allow the reaction to proceed; and fluorescent orenzymatic procedures to make evident this reaction.

[0246] In this preferred embodiment of the diagnosis kit, the cellsamples coming from the ill individual are made react with anti-lipidicparticles monoclonal antibody, in other words, with the indicatorreagent.

[0247] In an alternative embodiment of the diagnosis kit, instead of theanti-lipidic particles monoclonal antibody, it can be used at least apatient serum in which anti-lipidic particles antibodies have beenpreviously demonstrated by using the methodology described in Examples 1and 2B.

[0248] In an alternative embodiment, the diagnosis kit includes as somepart of it one or more microtiter plate(s) for cellular culture, orcentrifuge tube(s) as recipient(s) for the development of the reaction.

[0249] In another preferred embodiment of the present invention, a kitfor the detection of lipidic particles in cells in different physiologicstates coming from a human or animal subject, includes: at least anindicator reagent including at least an anti-lipidic particlesmonoclonal antibody; at least a buffer solution as a medium to allow thereaction; and fluorescent or enzymatic procedures to make evident thisreaction.

[0250] In this preferred embodiment of the detection kit, cell samplesin different physiologic states are made react with the anti-lipidicparticles monoclonal antibody, in other words, with the indicatorreagent.

[0251] In an alternative embodiment, the kit of detection of lipidicparticles in cells in different physiologic states includes as some partof it one or more microtiter plate(s) for cellular culture, orcentrifuge tube(s) as recipient(s) for the development of the reaction.

Example 4 Obtention of Mice that Produce Anti-Lipidic ParticlesAntibodies by Immunization With Liposomes Bearing Lipidic ParticlesInduced by Manganese

[0252] Ten, 2-months age, BALB/c female mice were immunized byintrasplenic injection of 100 μg of egg-yolkphosphatidylcholine:phosphatidate liposomes (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with MnCl₂ 5 mM to induce lipidic particlesformation. Intrasplenic immunization was repeated 2-weeks later by themethod described by Nilsson et al. (op. cit. 1987). Additionally, BALB/cfemale mice were intraperitoneally injected with the same liposomes dose2-weeks later, then they were boosted 4-times at 2-weeks intervals.

[0253] After seven days of the last immunization female mice were bledfrom the orbital sinus to analyze the presence of anti-lipidic particlesantibodies in the obtained sera. Using this immunization procedure, 60%of the immunized BALB/c female mice produced anti-lipidic particlesantibodies.

[0254] Immunoreaction analysis of mice sera was made by the liposomalcytofluorometry method. Egg-yolk phosphatidylcholine:phosphatidateliposomes (2:1 molar ratio) in Tris-NaCl buffer (10 mM, 1 mM) pH 7,containing 0.1 μmol of phosphatidate, and treated with 5 mM MnCl₂ toinduce lipidic particles formation were used as antigens. Analysis of10,000 liposomas was made in logarithmic mode with the followingdetectors: FSC in E00, with a detector compensation threshold of 52V;SSC of 401 V and FL-1 of 748 V (Baeza et al., op. cit., 1995). Theobtained data were analyzed with the Cellquest program (BecktonDickinson).

[0255] Autofluorescence and lipidic bilayer complexity (SSC) ofliposomes treated with MnCl₂ (FIG. 8A) were not modify when theseliposomes were also incubated with FITC-conjugated goat anti-Fc of miceIgG, IgA and IgM antibodies as second antibody (FIG. 8B). Because inabsence of anti-lipidic particles antibodies the second antibody do notbind directly to liposomes.

[0256] Mice sera were incubated with liposomal antigens and to detectthe immunoreaction they were used FITC-conjugated goat anti-Fc of miceIgG, IgA and IgM antibodies as second antibody. Sera analyzed wereobtained before mice were immunized as well as after the immunizationwith liposomal antigens.

[0257] Sera from mice before they were immunized did not show anyreaction with lipidic particles, since fluorescence and values of theirlipidic bilayer complexity (SSC) were similar to those of controlliposomes that were treated only with manganese (FIG. 8A). Sera weremixed and they were used as a negative control of mice seraimmunoreaction with lipidic particles (FIG. 8C).

[0258] Sera from mice after they were immunized with liposomal antigenstreated with manganese showed an immunoreaction that produce a liposomalfluorescence 10 to 100-fold higher than the reaction of mice controlsera (FIG. 8C), with values of D 2 0.5 at p<0.001. As example,cytofluorometry graphs of the reaction of serum from RB11, RB14 and RB17mice are showed in FIGS. 8D, 8E and 8F. Reaction between the antibodiesof sera from these mice and lipidic particles although positive, wasdifferent for each serum, with values of D=0.9, D=0.91 and D=0.79,respectively, which can be attributed to the polyclonal origin of theseantibodies. SSC values from immunoreaction (FIGS. 8D, 8E and 8F) weresimilar to those of liposomes control incubated with manganese (FIG.8A), and they showed the presence of lipidic particles which give thereaction with the anti-lipidic particles antibodies from mice sera.

[0259] Furthermore, the reaction of immunized mice sera with lipidicparticles did not show any liposomal aggregation, that could increase inunspecific way the fluorescence registered and to give a positive falseresult, since FSC values after the immunoreaction (FIGS. 9D-9F) weresimilar to those of liposomes incubated with manganese (FIG. 9A), orwith the second antibody (FIG. 9B), or with mice sera before theimmunization (FIG. 9C). SSC values in FIGS. 9A-9F also showed thepresence of lipidic particles in liposomal antigens as it was describedin FIGS. 8A-8F.

[0260] In mice immunized with liposomes bearing lipidic particlesinduced by manganese, after the detection of anti-lipidic particlesantibodies they were also detected anti-cardiolipin antibodies,anti-nuclear and anticoagulant antibodies. These findings confirm ourhypothesis which propose that anti-lipidic particles antibodiesconstitute the first stage in the development of illnesses associatedwith antiphospholipid antibodies. The mouse that gave the highestreaction with lipidic particles, was the RB14 with a value of D=0.91,and it was used in the obtention of anti-lipidic particles monoclonalantibodies.

Example 4A Obtention of Mice that Produce Anti-Lipidic ParticlesAntibodies by Immunization with Liposomes Bearing Lipidic ParticlesInduced by Chlorpromazine or Procainamide

[0261] Ten, 2-months age, BALB/c female mice were immunized byintrasplenic injection of 100 μg of egg-yolkphosphatidylcholine:phosphatidate liposomes (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with the lipidic particles inducer drugprocainamide at a concentration of 8 mM. Immunization was carried out asit was indicated in Example 4. After seven days of the last immunizationfemale mice were bled from the orbital sinus to analyze the presence ofanti-lipidic particles antibodies in the obtained sera. Using thisimmunization procedure, 70% of the immunized BALB/c female mice producedanti-lipidic particles antibodies.

[0262] Immunoreaction analysis of mice sera was made by the liposomalcytofluorometry method as it was indicated in Example 4. Egg-yolkphosphatidylcholine:phosphatidate liposomes (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 8 mM procainamide to induce lipidicparticles formation were used as antigens. Analysis of 10,000 liposomaswas made in logarithmic mode as it was described in Example 4.

[0263] Autofluorescence and lipidic bilayer complexity (SSC) ofliposomes treated with procainamide (FIG. 10A) were not modify whenthese liposomes were also incubated with FITC-conjugated goat anti-Fc ofmice IgG, IgA and IgM antibodies as second antibody (FIG. 10B). Becausein absence of anti-lipidic particles antibodies the second antibody donot bind directly to liposomes bearing lipidic particles induced byprocainamide.

[0264] Mice sera were incubated with liposomal antigens and to detectthe immunoreaction they were used FITC-conjugated goat anti-Fc of miceIgG, IgA and IgM antibodies as second antibody. Sera analyzed wereobtained before mice were immunized as well as after the immunizationwith liposomal antigens bearing lipidic particles induced byprocainamide.

[0265] Sera obtained before mice were immunized did not show anyreaction with lipidic particles, since fluorescence and values of theirlipidic bilayer complexity (SSC) were similar to those of controlliposomes that were treated only with procainamide (FIG. 10A). Seraobtained before mice immunization were mixed and the mixture was used asa negative control of mice sera immunoreaction with lipidic particles(FIG. 10C).

[0266] Sera obtained after mice were immunized with liposomal antigenstreated with the lipidic particles inducer drug procainamide showed animmunoreaction that produce a liposomal fluorescence 10 to 100-foldhigher than the reaction of control mice sera (FIG. 10C), with values ofD≧0.5 at p<0.001. As example, cytofluorometry graphs of the reaction ofserum from RF11, RF14 and RF17 mice are showed in FIGS. 10D, 10E and10F, respectively. Reaction between the antibodies of sera from thesemice and lipidic particles although positive, was different for eachserum, with values of D=0.8, D=0.72 and D=0.67, respectively, which canbe attributed to the polyclonal origin of these antibodies. SSC valuesfrom immunoreaction (FIGS. 10D, 10E and 10F) were similar to those ofliposomes control incubated with procainamide (FIG. 10A), and theyshowed the presence of lipidic particles in liposomes which give thereaction with the anti-lipidic particles antibodies from mice sera.

[0267] Furthermore, the reaction of sera from immunized mice did notproduce any liposomal aggregation, that could increase in unspecific waythe fluorescence registered and to give a positive false result, sinceFSC values after the immunoreaction were similar to those of liposomesincubated with procainamide, or with the second antibody, or with micesera before the immunization in a similar way as it was described inFIGS. 9A-9F.

[0268] Similar results to those shown in FIGS. 10A-10F were obtainedwhen mice were immunized with liposomes bearing lipidic particlesinduced by the lipidic particles inductor drug chlorpromazine at aconcentration of 3 mM.

[0269] After the detection of anti-lipidic particles antibodies inimmunized mice anti-cardiolipin antibodies, anti-nuclear andanticoagulant antibodies were also detected in them, in a similarfashion as it was described for mice immunized with liposomes treatedwith manganese, from Example 4. These findings corroborate thatanti-lipidic particles antibodies constitute the first stage in thedevelopment of illnesses associated with antiphospholipid antibodies.Furthermore, in mice immunized with liposomes incubated withprocainamide or chlorpromazine it has been demonstrated the presence ofdeposits of immune complexes in different organs. In addition, thesemice developed alopecia and lesions in the face in the form of butterflywings similar to those that have been described in human systemic lupuserythematosus. In FIG. 11 the picture of a 7-months age BALB/c femalemouse that was treated with liposomes bearing lipidic particles inducedby chlorpromazine, where alopecia and lesions in the face in the form ofbutterfly wings can be observed.

[0270] This animal model indicates that antigens bearing lipidicparticles induced by chlorpromazine or procainamide were more efficientin developed in BALB/c female mice not only anti-lipidic particlesantibodies, but a pathology that is more similar to the one that ispresented in humans.

Example 4B Obtention of Mice that Produce Anti-Lipidic ParticlesAntibodies by Immunization With the Drugs Chlorpromazine or Procainamide

[0271] For this treatment the immunization procedure indicated inExample 4A was modified since chlorpromazine or procainamide drugs wereadministered directly in absence of liposomal antigens to BALB/c femalemice.

[0272] Ten, 2-months age, BALB/c female mice were immunized byintramuscular injection of the lipidic particles inducer drugschlorpromazine or procainamide, using 3 mg/Kg, of body weight, forchlorpromazine and 10 mg/Kg, of body weight, for procainamide, each 24hs, for 2-months. Drug doses were similar to those that are administeredin the medical treatment of humans; in psychotic and maniacsdysfunctions for chlorpromazine and those that were used for thetreatment of cardiac arrhythmias for procainamide.

[0273] After seven days of the last intramuscular injection female micewere bled from the orbital sinus to analyze the presence of anti-lipidicparticles antibodies in the obtained sera. Using this immunizationprocedure, 50% of the immunized BALB/c female mice produced anti-lipidicparticles antibodies.

[0274] Immunoreaction analysis of mice sera was made by the liposomalcytofluorometry method as it was indicated in Example 4. Egg-yolkphosphatidylcholine:phosphatidate liposomes (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 8 mM procainamide to induce lipidicparticles formation were used as antigens. Analysis of 10,000 liposomaswas made in logarithmic mode as it was described in Example 4.

[0275] Mice sera were incubated with liposomal antigens and to detectthe immunoreaction they were used FITC-conjugated goat anti-Fc of miceIgG, IgA and IgM antibodies as second antibody. Sera analyzed wereobtained before mice were treated with the lipidic particles inducerdrugs as well as after of these treatments.

[0276] Sera obtained from mice after they were treated with the lipidicparticles inducer drug procainamide showed an immunoreaction thatproduce a liposomal fluorescence 4-fold higher than the reaction of micesera before treatment (a, FIGS. 12A, D, and G), with values of D≧0.5 atp<0.001. As example, cytofluorometry graphs of the reaction of serumfrom RP37, RP38 and RP39 mice are showed in: b, FIG. 12A; g, FIG. 12D;and j, FIG. 12G. Reaction between the antibodies of sera from these miceand lipidic particles although positive, was different to each serum,with values of D=0.58, D=0.68 and D=0.8, respectively, which can beattributed to the polyclonal origin of these antibodies. SSC values fromimmunoreaction showed in: d, FIG. 12B; h, FIG. 12E; and k, FIG. 12H weresimilar to those of liposomes control incubated with procainamide (FIG.10A), and they showed the presence of lipidic particles which give thereaction with the anti-lipidic particles antibodies from mice sera.

[0277] Furthermore, the reaction of immunized mice sera with lipidicparticles did not show any liposomal aggregation, since FSC values afterthe immunoreaction (f, FIG. 12C; i, FIG. 12F; and l, FIG. 12I) weresimilar to those of liposomes incubated with manganese (FIG. 9, Example4), or with mice sera before mice were treated with the lipidicparticles inducer drug procainamide (e, FIGS. 12C, F and I).

[0278] Anti-lipidic particles antibodies were also detected before thananti-cardiolipin antibodies, anti-nuclear and anticoagulant antibodiesin these mice, in a similar way as it was described for mice in Examples4 and 4A. Furthermore, the presence of deposits of immune complexes indifferent organs and the development of alopecia and lesions in the facein the form of butterfly wings were also showed in these mice.

[0279] Similar results to those shown in FIGS. 12A-12I were obtainedwhen BALB/c female mice were treated with the lipidic particles inducerdrug chlorpromazine.

[0280] These results indicate that the lipidic particles inducer drugschlorpromazine or procainamide induce the formation of lipidic particlesin the membranes of mice cells which subsequently induce the productionof anti-lipidic particles antibodies and the development of a pathologysimilar to the human antiphospholipid syndrome secondary to systemiclupus erythematosus. The formation of lipidic particles by the lipidicparticles inducer drugs chlorpromazine or procainamide in liposomes hasbeen previously demonstrated by nuclear magnetic resonance (Baeza etal., op. cit., 1995; Aguilar, op. cit., 1997; Aguilar et al., op. cit.,1999).

Example 4C Obtention of Producing Anti-Lipidic Particles Antibodies Miceby Passive Immunization With the Anti-Lipidic Particles H308 MonoclonalAntibody

[0281] For this treatment the immunization procedure indicated inExample 4 was modified since passive immunization of BALB/c female micewas carried out.

[0282] Ten, 2-months age, BALB/c female mice were immunized byintraperitoneal injection of 1 μg of anti-lipidic particles H308monoclonal antibody, each week, for 2-months. After seven days of thelast intraperitoneal injection female mice were bled from the orbitalsinus to analyze the presence of anti-lipidic particles antibodies inthe obtained sera. Using this immunization procedure, 80% of theimmunized BALB/c female mice produced anti-lipidic particles antibodies.

[0283] Immunoreaction analysis of mice sera was made by the liposomalcytofluorometry method as it was indicated in Example 4. Egg-yolkphosphatidylcholine:phosphatidate liposomes (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 5 mM CaCl₂ to induce lipidic particlesformation were used as antigens. Analysis of 10,000 liposomas was madein logarithmic mode as it was described in Example 4.

[0284] Mice sera were incubated with liposomal antigens and to detectthe immunoreaction they were used FITC-conjugated goat anti-Fc of miceIgG, IgA and IgM antibodies as second antibody. Sera analyzed wereobtained before mice were treated by passive immunization with theanti-lipidic particles H308 monoclonal antibody as well as after micereceived this immunization procedure.

[0285] Sera obtained after mice were treated by passive immunizationwith the anti-lipidic particles H308 monoclonal antibody showed thepresence of anti-lipidic particles antibodies in them; sincecytofluorometry graphs of the reaction of passive immune mice sera withliposomes bearing lipidic particles induced by CaCl₂ were similar tothose described in FIGS. 8, 9, 10 and 12 which showed anti-lipidicparticles antibodies induced by different antigens containing lipidicparticles.

[0286] Anti-lipidic particles antibodies were also detected before thananti-cardiolipin antibodies, anti-nuclear and anticoagulant antibodiesin these mice, in a similar way as it was described for mice in Examples4, 4A, and 4B. Additionally, the presence of deposits of immunecomplexes in different organs and the development of alopecia andlesions in the face in the form of butterfly wings were also showed inthese mice.

[0287] These results showed the direct participation of anti-lipidicparticles antibodies in the development in BALB/c female mice of apathology similar to the human antiphospholipid syndrome secondary tosystemic lupus erythematosus. Therefore, a possible treatment of theseillnesses would be by the inhibition of anti-lipidic particlesantibodies and/or by the stabilization of cellular membranes thatprevent the formation of lipidic particles, as it is subsequentlydescribed.

Example 5 Obtention of Hybridomas by Fusion of P3X63Ag8U.1 Cells WithSpleen Cells of a Producing Anti-Lipidic Particles Antibodies BALB/cFemale Mouse

[0288] Four days before the planned fusion, three mice previouslyimmunized by intrasplenic and intraperitoneal injection of 100 μl ofegg-yolk phosphatidylcholine:phosphatidate liposomes (2:1 molar ratio)in Tris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 5 mM MnCl₂ to induce lipidic particlesformation, were boosted by intravenous tail vein injection using thesame liposomes dose. The rational way is to initiate secondary immuneresponses selectively in the spleen as opposed to lymph nodes. Thereforethe mouse producing the higher titer of anti-lipidic particlesantibodies were chosen for hybridoma production.

[0289] The spleen of the RB14 BALB/c female mouse producing the highertiters of anti-lipidic particles antibodies was removed under sterilityconditions and it was placed in a petri dish with 6 ml of incompleteDMEM cell culture medium. Spleen mouse was dispersed until a suspensionof single cells was obtaining using blunt tips pincers. Cellularsuspension was transferred to a 15-ml falcon tube and it was left inrepose so that the thick residuals settle down. Next, cellularsuspension was transferred to another falcon tube and it was centrifugedat 17×g for 7 min. Subsequently the supernatant was decanted andcellular pellet was resuspended by gently agitation and cellularsuspension was diluted by the addition, drop by drop, of 10 ml ofincomplete DMEM cell culture medium. Cellular suspension was centrifugedas it was already indicated, then the supernatant was decanted and 4 mlof 0.16 M NH₄Cl were added for erythrocytes lysis. In this step the tubecontaining cellular suspension was incubated at 37° C. and it was gentlyrotated during 4 min. Later on 6 ml of incomplete DMEM cell culturemedium was added and cellular suspension was centrifuged at 17×g for 7min. After centrifugation the supernatant was decanted and cellularpellet was gently resuspended in 10 ml of incomplete DMEM cell cultureand was allowed to stand at room temperature until their were used(Kohler and Milstein, 1975. Nature 256:495.497).

[0290] On the other hand, P3x63Ag8U.1 myeloma cells were collected fromcell culture plates and transferred to falcon tubes. Aliquots fromP3x63Ag8U.1 myeloma cells and mouse spleen cells were treated withtrypan blue and they were counted in a Neubauer camera. The viability ofboth cellular suspension were higher than 95%. P3x63Ag8U.1 myeloma cellsand mouse spleen cells were mixed in a 1:1 cellular proportion, using36×10⁶ cells of each cellular type, later cellular mixture was washedwith 10 ml of incomplete DMEM cell culture medium. After centrifugationat 17 ×g for 5 min the supernatant was decanted and cellular pellet wasgently resuspended. Subsequently, 1 ml of 4000 polyethyleneglycolsolution was added drop by drop, during 1 min, and the mixture wasmanually shaken up for 1.5 min, then 1 ml of incomplete DMEM cellculture medium was added for 30 sec with slow tube rotation. Next, 3 mlof incomplete DMEM cell culture medium was added for 30 sec also withslow tube rotation, later 16 ml of the same medium was added for 1.5 minwith gently agitation. Finally the volume of the fused cell suspensionwas completed to 40 ml with incomplete DMEM cell culture medium andfused cell suspension was incubated without agitation for 5 min at roomtemperature. Later on fused cell suspension was centrifuged at 17×g for5 min, the supernatant was decanted and fused cell pellet was washedagain with 40 ml of incomplete DMEM medium. Fused cell pellet wasresuspended in 30 ml of selection DMEM-HAT medium and aliquots of 100 μlof this fused cell suspension were seeded in each one of the wells ofthree 96-wells flat-bottom microtiter plates which 24 hs before cellfusion were seeded with macrophages as feeder cells. Microtiter plateswere incubated at 37° C., in an atmosphere with 5% of CO₂. After five oreight days of the cellular fusion hybridomas were fed with 50 μl ofselection DMEM-HAT medium, finally after 11 days of the cellular fusionhybridoma supernatants were changed by 100 μl of DMEM-HAT media.

[0291] After hybridomas growing the supernatants were screened by theliposomal-ELISA method in order to detect the production of anti-lipidicparticles antibodies by them. Cellular samples from all hybridomasproducing anti-lipidic particles monoclonal antibodies were frozen at−70° C. in liquid nitrogen. Later, 10 hybridomas with high anti-lipidicparticles monoclonal antibodies titers were chosen (Table 3) and theywere cloned again by limiting dilution in 96-wells flat-bottommicrotiter plates. After hybridomas growing supernatants were screenedagain by the liposomal-ELISA method and those producing the highertiters of anti-lipidic particles monoclonal antibodies were cultivatedin 250 ml bottles for the massive obtention of supernatants containingthese antibodies. TABLE 3 Hybridomas producing anti-lipidic particlesantibodies. Hybridoma Arbitrary Units Hybridoma Arbitrary Units H40 26H120 36 H65 32 H121 35 H70  3 H176 42 H90 22 H200 30  H110 23 H308 48

Example 6 Detection of the Inhibition of Anti-Lipidic Particles H308Monoclonal Antibody Using Phosphorylated Hastens by the Liposomal-ELISAMethod

[0292] Costar microtiter plates, with 96 flat-bottom wells with a highlipidic antigens binding property (Costar Co. Cambrige, USA) were coatedby the addition to each one of the wells of 100 μl of liposomes madefrom egg-yolk phosphatidylcholine:phosphatidate (2:1 molar ratio) inTris-NaCl buffer (10 mM; 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 5 mM CaCl₂ to induce lipidic particlesformation. Microtiter plates were incubated at room temperature for 1 hand they were blocked for 1 h at room temperature in a similar way aswas described in Example 1. Next, blocking solution was discarded bysuction and 100 μl of H308 monoclonal antibody that was previouslyincubated with the phosphorylated haptens were added immediately to eachone of the wells, to avoid that they becomes dry off.

[0293] Phosphorylcholine, glycerolphosphorylcholine, phosphorylserine,glycerol-phosphorylserine and phosphorylethanolamine were used ashaptens in quantities of 0.2, 0.4, 0.6, 0.8 and 1.0 μmol. The chemicalstructure of these haptens is presented in FIG. 13. Phosphorylcholineand glycerolphosphorylcholine constitute part of the polar region of thelipid phosphatidylcholine, as well as phosphorylserine andglycerolphosphorylserine constitute part of the polar region ofphosphatidylserine, and phosphorylethanolamine is part ofphosphatidylethanolamine.

[0294] Aliquots of 100 μl of H308 monoclonal antibody were incubatedwith 100 μl of each one of the hapten solutions for 30 min at 30° C.Later on, the liposomal-ELISA method was applied as it was described inExample 1. Peroxidase-conjugated goat anti-Fc of mouse IgM antibodieswere used as second antibody.

[0295] When phosphorylcholine was used as hapten to blocking H308monoclonal antibody a decrease in the immunoreaction of this antibodywith liposomal antigens bearing lipidic particles was observed. Thisdecrease gave an absorbance at 492 nm of 0.06, with 0.6 μmoles of hapten(E, FIG. 14) which represent a 82% inhibition in immunoreaction withregard to reaction in absence of hapten (A, FIG. 14). With 0.2 μmoles ofglycerol-phosphorylcholine an 100% inhibition of H308 monoclonalantibody was showed (F, FIG. 14). On the other hand,glycerolphosphorylserine (B, FIG. 14), phosphorylserine (C, FIG. 14) andphosphorylethanolamine (D, FIG. 14) do not cause any inhibition of H308monoclonal antibody reaction with lipidic particles in liposomes.

[0296] Inhibition of H308 monoclonal antibody reaction withphosphorylcholine and glycerolphosphorylcholine indicate that theantigen recognition domain in H308 monoclonal antibody has subdomainsthat recognize specifically the choline methyl groups which lacksethanolamine and serine (FIG. 13). In addition, total immunoreactioninhibition attained by glycerolphosphorylcholine suggests that theantigen domain that recognize H308 monoclonal antibody include chemicalgroups of glycerol. These findings are in agreement with the structuralpattern proposed for the lipidic particle (Cullis et al., op. cit.,1991) (FIG. 15) where monolayer lipids (C, FIG. 15) that recover themolecular arrangement different to bilayer (B, FIG. 15) are moreseparate than lipids that constitute a normal monolayer (A, FIG. 15). Inan open monolayer (C, FIG. 15) glycerolphosphorylcholine is more exposedthan in a normal bilayer therefore this is the region in which the H308monoclonal antibody reacts.

[0297] Possibly the central domain of lipidic particle, the region thatis observed as inverted micella in: B, FIG. 15, is formed by conicshaped lipids such as phosphatidate. In contrast, monolayers most openthan a normal monolayer would be formed by phosphatidylcholine and theywould be the regions that identify the H308 monoclonal antibody. If H308monoclonal antibody reacts specifically with a phosphatidylcholine openmonolayer, is clear that this antibody does not show any immunoreactionwith liposomes formed exclusively by phosphatidylcholine (G, FIG. 14),because in these liposomes the lipids are in a normal monolayerassociation that constitute the bilayer, in consequence noimmunoreaction with H308 monoclonal antibody is detected.

Example 6A Detection by the Liposomal-ELISA Method ofGlycerolphosphorylcholine Hapten Inhibition of Anti-Lipidic ParticlesAntibodies from Sera of Patients With the Antiphospholipid Syndrome

[0298] Costar microtiter plates, with 96 flat-bottom wells with a highlipidic antigens binding property (Costar Co. Cambrige, USA) were coatedby the addition to each one of the wells of 100 μl of liposomes madefrom egg-yolk phosphatidylcholine:phosphatidate (2:1 molar ratio) inTris-NaCl buffer (10 mM, 1 mM) pH 7, containing 0.1 μmol ofphosphatidate, and treated with 5 mM CaCl₂ to induce lipidic particlesformation.

[0299] Aliquots of 100 μl of patients' sera that were analyzed inExamples 1 and 2B were incubated with 100 μl of 0.2 μmoles ofglycerolphosphorylcholine for 30 min at 30° C. Later on, blockedpatients sera were added to the wells of the microtiter plate and theliposomal-ELISA method was applied as it was described in Example 1.Peroxidase-conjugated goat anti-Fc of human IgG, IgA and IgM antibodieswere used as second antibody.

[0300] Gycerolphosphorylcholine hapten at a concentration of 0.2 μmolesproduce an 100% inhibition in the immunoreaction of patients sera withlipidic particles in liposomes, in a similar way as it was described bythe inhibition of H308 monoclonal antibody in FIG. 14 graphs, Example 6.

[0301] These results confirm that sera from patients with theantiphospholipid syndrome have anti-lipidic particles antibodies with anantigenic specificity similar to that of H308 monoclonal antibody, sincethey were inhibited in the same proportion by thegycerolphosphorylcholine hapten.

[0302] Studies in BALB/c female mice in which were simultaneouslyadministered the H308 monoclonal antibody, which developed in these micea pathology similar to human antiphospholipid syndrome as it wasdescribed in Example 4C, and the glycerol-phosphorylcholine hapten,showed a blockage in the development of the pathology in BALB/c femalemice. H308 monoclonal antibody was administered by intraperitonealinjection of 1 μg each week during two months to BALB/c female mice, andsimultaneously the glycerolphosphorylcholine hapten was administered at2.5 mg/Kg, of body weight, doses by intravenous injection each 24 hs,for 2-months. With this treatment it was inhibited in 40% thedevelopment of mice pathology by H308 monoclonal antibody.

[0303] In accordance with the above-mentioned studies thetherapeutically effective quantity of the inhibitor drugglycerolphosphorylcholine is of 2.5 mg/Kg of body weight.

Example 7 Study by the Liposomal Cytofluorometry Method of theStabilization of Liposomal Membranes that Prevent the Formation ofLipidic Particles and the Subsequent Binding of Anti-Lipidic ParticlesAntibodies

[0304] These studies were carried out with a modification in theliposomal cytofluorometry method with liposomes made from egg-yolkphosphatidylcholine:phosphatidate (2:1 molar ratio) in Tris-NaCl buffer(10 mM, 1 mM) pH 7, containing 0.1 μmol of phosphatidate, and treatedwith 0.2 mM chlorpromazine to induce lipidic particles formation, asantigens.

[0305] Immediately after the addition of the lipidic particles inducerdrug chlorpromazine liposomes were incubated with differentconcentrations of the lipid bilayer stabilizers drugs spermidine orchloroquine. Liposomal preparations were incubated for 30 min at roomtemperature and were used as antigens. The reaction of these stabilizedliposomes with H308 monoclonal antibody was analyzed by the liposomalcytofluorometry method as was described in Example 2A, in a FACSCaliburFlow Cytometer equipped with a single 488 nm argon laser beam (BecktonDickinson).

[0306] Relative size and/or liposomal aggregation were analyzed in theFSC channel and the granularity or liposomal bilayers complexity in theSSC channel. Analysis of 10,000 liposomes was made in a logarithmic modewith the following detectors: FSC in E00, with a detector compensationthreshold of 52 V and SSC of 401 V (Baeza et al., op. cit., 1995). Theobtained data were analyzed with the Cellquest program (BecktonDickinson).

[0307] When egg-yolk phosphatidylcholine:phosphatidate (2:1 molar ratio)liposomes, in Tris-NaCl buffer (10 mM, 1 mM) pH 7, and containing 0.1μmol of phosphatidate (A, FIG. 16), were treated with 0.2 mMchlorpromazine an 100-fold increase in SSC and FSC values were observedwhich showed the presence of lipidic particles and liposomalaggregation, respectively (B, FIG. 16).

[0308] In contrast, the incubation of egg-yolkphosphatidylcholine:phosphatidate (2:1 molar ratio) liposomes, inTris-NaCl buffer (10 mM, 1 mM) pH 7, and containing 0.1 μmol ofphosphatidate, with the stabilizer drugs spermidine (C, FIG. 16) orchloroquine (D, FIG. 16) do not produce any change in liposomalaggregation and in liposomal bilayers complexity because the graphsobtained were similar to that corresponding to liposomes in buffersolution (A, FIG. 16). However, when liposomes were incubatedsimultaneously with the lipidic particles inducer drug chlorpromazineand the lipidic bilayer stabilizer drugs spermidine (E, F, FIG. 16) orchloroquine (G, H, FIG. 16) there were not lipidic particles formationneither liposomal aggregation. It can be observed that SSC and FSCvalues (E-H, FIG. 16) were very similar to those of liposomes in buffersolution, which showed SSC values smaller than 100 units; contrary tothe graph that indicates the presence of lipidic particles, with SSCvalues higher than 1000 units (B, FIG. 16).

[0309] These studies showed that spermidine is effective in stabilizinglipidic bilayers at concentrations 5 μM, this spermidine quantityblockage the formation of lipidic particles induced by chlorpromazine atconcentrations of 0.2 mM and 0.6 mM, respectively (E, F, FIG. 16). Forchloroquine, effective concentrations were even smaller, since thisstabilizer drug produced liposomal stabilization at a concentration of0.1 μM when the lipidic particles inducer drug chlorpromazine were usedat 0.2 mM and 0.6 mM, respectively (G, H, FIG. 16).

[0310] When liposomes incubated with both drugs: the lipidic particlesinducer drug and the lipidic bilayer stabilizer drug were used asantigens there was not any immunoreaction with the H308 monoclonalantibody, because the cytofluorometry graphs obtained were as thosecorresponding to liposomes alone and in absence of antibodies in: g,FIG. 3D; and i, FIG. 3E, instead of as those that indicate the reactionof H308 monoclonal antibody with lipidic particles in: b, FIG. 3A; andd, FIG. 3B.

[0311] These results indicate that liposomal membranes were stabilizedby their interaction with lipidic bilayer stabilizers drugs spermidineor chloroquine in consequence they do not contain lipidic particles andtherefore they do not react with H308 monoclonal antibody.

Example 7A Study by the Cytofluorometry Method of the Cellular MembranesStabilization that Prevents the Formation of Lipidic Particles and theLater Binding of Anti-Lipidic Particles Antibodies

[0312] Ag4 mouse myeloma cells suspended in Tris-NaCl buffer (10 mM, 135mM) pH 7, containing glucose 11 mM, were incubated with the lipidicparticles inducer drug chlorpromazine 0.2 mM. Immediately after theaddition of chlorpromazine the Ag4 mouse myeloma cells were incubatedwith different concentrations of the lipid bilayer stabilizer drugsspermidine or chloroquine for 30 min at room temperature. The reactionof these stabilized Ag4 mouse myeloma cells with H308 monoclonalantibody was analyzed by the cytofluorometry method in a FACSCaliburFlow Cytometer equipped with a single 488 nm argon laser beam (BecktonDickinson).

[0313] Relative size and/or Ag4 mouse myeloma cells aggregation wereanalyzed in the FSC channel and the granularity or cellular membranescomplexity in the SSC channel. Analysis of 10,000 Ag8 mouse myelomacells was made with the following detectors: FSC in E00 in lineal modewith an amplifier gain of 2 V, with a detector compensation threshold of52 V, and SSC of 250 V. The obtained data were analyzed with theCellquest program (Beckton Dickinson).

[0314] Results obtained with Ag4 mouse myeloma cells incubated with thelipidic particles inducer drug chlorpromazine and the lipid bilayerstabilizer drugs spermidine or chloroquine were similar to thosedescribed in FIG. 16 graphs. These results showed that Ag4 cellularmembranes were stabilized by their interaction with lipidic bilayerstabilizers drugs spermidine or chloroquine in consequence they do notdevelop lipidic particles with chlorpromazine.

[0315] When Ag4 mouse myeloma cells incubated with both drugs: thelipidic particles inducer drug and the lipidic bilayer stabilizer drugwere used as antigens there were not any immunoreaction with the H308monoclonal antibody, in a similar way as was demonstrated for liposomesstabilized with the drugs spermidine or chloroquine in Example 7.

[0316] These results indicate that cellular membranes of Ag4 mousemyeloma cells stabilized by their interaction with spermidine orchloroquine do not develop lipidic particles and therefore they do notreact with the H308 monoclonal antibody.

Example 7B Detection of the Lipidic Particles Reversion by the LipidicBilayer Stabilizer Drugs Spermidine and Chloroguine

[0317] Examples 7 and 7A were repeated using as antigens egg-yolkphosphatidylcholine: phosphatidate (2:1 molar ratio) liposomes, inTris-NaCl buffer (10 mM, 1 mM) pH 7, and containing 0.1 μmol ofphosphatidate, or Ag4 mouse myeloma cells. Liposomal or cellularantigens were incubated with the lipidic particles inducer drugchlorpromazine 0.2 mM for 30 min at room temperature before thetreatment with the lipidic bilayers stabilizer drugs spermidine orchloroquine. Stabilizer drugs were used at the concentrations used inExample 7A.

[0318] Cytofluorometric analysis showed that the formation of lipidicparticles and the liposomal or cellular aggregation caused bychlorpromazine, which produce graphics similar to: B, FIG. 16, werereverted by the lipid bilayer stabilizers spermidine or chloroquine.This reversion that destroy lipidic particles in liposomal and cellularantigens produce graphics as those shown in: E, F, G, and H, FIG. 16,which showed lipids in bilayer molecular arrangements.

[0319] After lipidic particles reversion liposomal or cellular antigensdo not show any reaction with H308 monoclonal antibody, due to theabsence of lipidic particles in their surfaces.

[0320] These experiments showed that spermidine and chloroquine drugshave the action of prevent the formation of lipidic particles andadditionally they can also destroy lipidic particles already formed.These findings are very important for the application of thesestabilizer drugs in the treatment of human illnesses in which lipidicparticles and/or anti-lipidic particles antibodies participate.

[0321] Studies in BALB/c female mice in which were simultaneouslyadministered the H308 monoclonal antibody, which develop in these mice apathology similar to human antiphospholipid syndrome as it was describedin Example 4C, and one of the lipid bilayer stabilizer drugs spermidineor chloroquine, showed a delay in the development of this pathology.H308 monoclonal antibody was administered by intraperitoneal injectionof 1 μg each week for 2-months to BALB/c female mice, and simultaneouslythe stabilizer drug chloroquine was administered at 2.5 mg/Kg, of bodyweight, oral doses each 24 hs, during two months. With this treatment itwas delayed the development of mice pathology induced by H308 monoclonalantibody. It is possible that modifying the doses of stabilizers drugsthat are applied to mice it is possible to prevent the development ofthese illnesses. The chloroquine dose used was similar to the one usedin humans in the treatment of rheumatoid arthritis and of systemic lupuserythematosus.

[0322] Spermidine was administered, as chlorhydrate, in a dose of 1mg/Kg, of body weight, by intraperitoneal injection each 24 hs, duringtwo months. Using this spermidine dose results similar to thosedescribed with chloroquine were obtained.

[0323] According to the above-mentioned studies the therapeuticallyeffective quantity of the lipidic bilayer stabilizer drug chloroquine isof 2.5 mg/Kg, of body weight, and of spermidine is of 1 mg/Kg, of bodyweight.

[0324] In accordance with the information described in this work, onewill be able to observe that the use of antibodies obtained by usinglipidic structures different to lipid bilayers to determine cellularphysiologic states and for the diagnosis and/or treatment of diseaseshas been designed to allow an early detection of diseases associatedwith antiphospholipid antibodies and, as a consequence, to allow atreatment to prevent, to stop and to revert this disease; and it will beevident for any expert in the matter that the modalities that here arepresented, they are only illustrative and they will not be interpretedin a limitative form of the present invention, since there are possibleother numerous changes in their details and particularities, withoutmoving away from the scope of the invention.

[0325] Though it has been illustrated and described specific embodimentsof the invention, it is necessary to emphasize that are possible othernumerous modifications to the invention, as they can be the use ofdifferent mice strains, lipids to obtain the liposomes, immunizationmethods and methods for obtaining hybridomas, diverse reagents for thekit of diagnosis and/or diverse illnesses associated withantiphospholipid antibodies. Therefore, the present invention should notbe considered as restricted except for which demands the previoustechnique and for the spirit of the annexed claims.

What is claimed:
 1. A kit for use in an assay to indirectly determinethe presence of lipidic particles in cell membranes from a samplesuspected of having anti-lipidic particle antibodies from an individualsuspected of suffering primary antiphospholipid syndrome or one diseaseassociated with secondary antiphospholipid syndrome, wherein thepresence of said lipidic particles in cell membranes allows diagnosis ofwhether said individual is developing an illness associated with thepresence of antiphospholipid antibodies though said individual does notpresent anti-cardiolipin antibodies, lupus anti-coagulant, anti-DNAantibodies or anti-nuclear antibodies; comprising: a) an indicatorreagent comprising an antigen having lipidic particles to be contactedwith the sample from said individual under conditions effective topermit binding of anti-lipidic particle antibodies present in the sampleto the lipidic particles of the antigen, wherein said sample from saidindividual does not present anti-cardiolipin antibodies, lupusanti-coagulant, anti-DNA antibodies or anti-nuclear antibodies; b) abuffer solution as a medium to allow effective conditions for thebinding of the anti-lipidic particle antibodies present in the sample tothe lipidic particles of the antigen; and c) a detectable-labeledreagent useful for detecting the binding of anti-lipidic particleantibodies present in the sample to the lipidic particles of theantigen, wherein the presence of the anti-lipidic particle antibodies inthe sample can be correlated with immune damage in cell membranes havinglipidic particles of said individual as one of the first events inillness associated with the presence of antiphospholipid antibodies. 2.The kit of claim 1, wherein said antigen comprises liposomes havinglipidic particles induced with one agent selected from the groupconsisting of divalent cations and drugs producing lupus in humans, andwherein said liposomes are in one condition selected from the groupconsisting of liposomes bound to microtiter plates with a high lipidicbinding property and liposomes suspended in an appropriate medium. 3.The kit of claim 1, wherein said antigen comprises neoplastic cellsbound to one solid support selected from the group consisting of microcover glasses and microtiter plates.
 4. The kit of claim 1, wherein thebuffer solution has a pH of 7.0 to 7.4.
 5. The kit of claim 1, whereinthe sample is selected from the group consisting of serum and plasma ofsaid individual.
 6. The kit of claim 1, wherein the detectable-labeledreagent comprises detectable-labeled polyvalent anti-humanimmunoglobulin second antibodies which bind to the anti-lipidic particleantibodies.
 7. The kit of claim 6, wherein the detectable-labeledanti-human immunoglobulin second antibodies comprise at least oneanti-human immunoglobulin antibody directed against at least one humanimmunoglobulin class and the presence of anti-lipidic particleantibodies is determined as an antibody selected from the groupconsisting of anti-lipidic particles IgG, IgM and IgA antibodies.
 8. Thekit of claim 1, wherein the detectable-labeled reagent comprises onecomponent selected from the group consisting of enzymes andfluorochromes, said component being attached to one element selectedfrom the group consisting of polyvalent anti-immunoglobulins, anti-IgG,IgM and IgA immunoglobulin second antibodies, and where the enzyme isselected from the group consisting of alkaline phosphatase andperoxidase, and the fluorochrome is selected from the group consistingof fluorescein isothiocyanate, phycoerythrin, Cy3 and Percp.
 9. The kitof claim 1, further including a blocking solution to prevent falsepositive results from occurring when microtiter plates are used as asolid support, and at least a sample of a reference serum from a healthyindividual as a negative control of the reaction with the antigencontaining lipidic particles.
 10. The kit of claim 1, further includingat least an anti-lipidic particle polyclonal or monoclonal antibody tobe reacted with the antigen having lipidic particles in order to confirmwhether the anti-lipidic particle antibodies are present or not in saidsample.
 11. The kit of claim 1, wherein the detection of the presence ofanti-lipidic particle antibodies in the sample from said individual iscarried out using the protocol selected from the group consisting ofliposomal-ELISA, cell-ELISA, immunofluorescence,liposomal-cytofluorometry and cell-cytofluorometry.
 12. The kit of claim1, wherein the antigen having lipidic particles is selected from thegroup consisting of liposomes, neoplastic cells, human erythrocytes,human leukocytes, and human plaquettes.
 13. The kit of claim 12, whereinsaid antigen is selected from the group consisting of erythrocytes,leukocytes, and plaquettes, and said antigen is suspended in a mediumconsisting of a buffer solution that allows effective conditions for thebinding of the anti-lipidic particle antibodies present in the sample tothe lipidic particles of the antigen.